901 Citations
We describe a fast easy and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide single-wavelength anomalous diffraction I-SAD The potential universality of the method is based on a common feature of membrane proteins the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins including a human G protein coupled receptor GPCR and we show that I-SAD can be applied using data collection strategies based on either standard or ... More
We describe a fast, easy, and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide–single-wavelength anomalous diffraction (I-SAD). The potential universality of the method is based on a common feature of membrane proteins—the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts. We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins, including a human G protein–coupled receptor (GPCR), and we show that I-SAD can be applied using data collection strategies based on either standard or serial x-ray crystallography techniques. Less
Lipidic cubic phase LCP has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment Application of this material to a wide variety of membrane proteins however is hindered due to a limited number of available host lipids mostly monoacylglycerols MAGs Here we designed synthesized and characterized a series of chemically stable lipids resistant to hydrolysis with properties complementary to the widely used MAGs In order to assess their potential to serve as host lipids for crystallization we characterized the phase properties and lattice parameters of mesophases made of ... More
Lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases made of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 °C and 20 °C. Less
Neisserial heparin-binding antigen NHBA is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero the crystal structures of two fragment antigen-binding domains Fabs isolated from human monoclonal antibodies targeting NHBA were determined Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs these structures provide broad insights into the NHBA epitopes recognized by the human immune system As expected these Fabs also show remarkable structural conservation ... More
Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA. Less
Second harmonic generation SHG was integrated with Raman spectroscopy for the analysis of pharmaceutical materials Particulate formulations of clopidogrel bisulphate were prepared in two crystal forms Form I and Form II Image analysis approaches enable automated identification of particles by bright field imaging followed by classification by SHG Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with confidence in a total measurement time of ms per particle Complementary measurements by Raman and synchrotron XRD are in excellent agreement with the classifications made by SHG with measurement times of minute and several seconds per particle respectively ... More
Second harmonic generation (SHG) was integrated with Raman spectroscopy for the
analysis of pharmaceutical materials. Particulate formulations of clopidogrel bisulphate were
prepared in two crystal forms (Form I and Form II). Image analysis approaches enable
automated identification of particles by bright field imaging, followed by classification by SHG.
Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with
99.95% confidence in a total measurement time of ~10 ms per particle. Complementary
measurements by Raman and synchrotron XRD are in excellent agreement with the
classifications made by SHG, with measurement times of ~1 minute and several seconds per
particle, respectively. Coupling these capabilities with at-line monitoring may enable real-time
feedback for reaction monitoring during pharmaceutical production to favor the more
bioavailable but metastable Form I with limits of detection in the ppm regime. Less
analysis of pharmaceutical materials. Particulate formulations of clopidogrel bisulphate were
prepared in two crystal forms (Form I and Form II). Image analysis approaches enable
automated identification of particles by bright field imaging, followed by classification by SHG.
Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with
99.95% confidence in a total measurement time of ~10 ms per particle. Complementary
measurements by Raman and synchrotron XRD are in excellent agreement with the
classifications made by SHG, with measurement times of ~1 minute and several seconds per
particle, respectively. Coupling these capabilities with at-line monitoring may enable real-time
feedback for reaction monitoring during pharmaceutical production to favor the more
bioavailable but metastable Form I with limits of detection in the ppm regime. Less
Biological molecules especially the proteins have a special and important function We study their structure to understand their functions and further make application like the medical research The routine method is diffraction but not work for molecules which cannot grow into crystal and molecules which their crystal are too small Cryo-EM technique provides another way to solve their structures through their images it does not need crystals Meanwhile electron diffraction can work small crystals micro- and nano-crystals after the sample preparation was improved Hence we try to build a method that can restore the structure from the crystal s image ... More
Biological molecules, especially the proteins, have a special and important function. We study their structure to understand their functions, and further make application, like the medical research. The routine method is diffraction, but not work for molecules which cannot grow into crystal and molecules which their crystal are too small. Cryo-EM technique provides another way to solve their structures through their images, it does not need crystals. Meanwhile, electron diffraction can work small crystals (micro- and nano-crystals) after the sample preparation was improved. Hence, we try to build a method that can restore the structure from the crystal�s image. We collect images of protein nano-crystal, and these images were processed to enhance their contrast. The key step is to find the orientations of these images in the procedure of reconstruction, therefore, we create a method that calculates these orientations. Nano-crystals, which cannot be used in diffraction method, then can be used in this method. Less
Structures of enzyme-substrate product complexes have been studied for over four decades but have been limited to either before or after a chemical reaction Recently using in crystallo catalysis combined with X-ray diffraction we have discovered that many enzymatic reactions in nucleic-acid metabolism require additional metal-ion cofactors that are not present in the substrate or product state By controlling metal ions essential for catalysis the in crystallo approach has revealed unprecedented details of reaction intermediates Here we present protocols used for successful studies of Mg -dependent DNA polymerases and ribonucleases that are applicable to analyses of a variety of metal ... More
Structures of enzyme-substrate/product complexes have been studied for over four decades but have been limited to either before or after a chemical reaction. Recently using in crystallo catalysis combined with X-ray diffraction, we have discovered that many enzymatic reactions in nucleic-acid metabolism require additional metal-ion cofactors that are not present in the substrate or product state. By controlling metal ions essential for catalysis, the in crystallo approach has revealed unprecedented details of reaction intermediates. Here we present protocols used for successful studies of Mg2+-dependent DNA polymerases and ribonucleases that are applicable to analyses of a variety of metal ion-dependent reactions. Less
Bacteria sense and adapt to environmental changes using two-component systems These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator RR which finally modulates the transcription of target genes The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response NtrX is a modular protein with REC AAA and DNA-binding domains an architecture that classifies it among the NtrC subfamily of RRs However it lacks the signature GAFTGA motif that is essential for activating transcription by ... More
Bacteria sense and adapt to environmental changes using two-component systems. These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator (RR), which finally modulates the transcription of target genes. The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response. NtrX is a modular protein with REC, AAA +, and DNA-binding domains, an architecture that classifies it among the NtrC subfamily of RRs. However, it lacks the signature GAFTGA motif that is essential for activating transcription by the mechanism proposed for canonical members of this subfamily. In this article, we present the first crystal structure of full-length NtrX, which is also the first structure of a full-length NtrC-like RR with all the domains solved, showing that the protein is structurally similar to other members of the subfamily. We also report that NtrX binds nucleotides and the structures of the protein bound to ATP and ADP. Despite binding ATP, NtrX does not have ATPase activity and does not form oligomers in response to phosphorylation or nucleotide binding. We also identify a nucleotide sequence recognized by NtrX that allows it to bind to a promoter region that regulates its own transcription and to establish a negative feedback mechanism to modulate its expression. Overall, this article provides a detailed description of the NtrX RR and supports that it functions by a mechanism different to classical NtrC-like RRs. Less
Autophagy is a conserved cellular process involved in the elimination of proteins and organelles It is also used to combat infection with pathogenic microbes The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg LC proteins coupled to phosphatidylethanolamine PE on autophagosomal membranes To understand how RavZ recognizes and deconjugates LC -PE we prepared semisynthetic LC proteins and elucidated the structures of the RavZ LC interaction Semisynthetic LC proteins allowed the analysis of structure-function relationships RavZ extracts LC -PE from the membrane before deconjugation RavZ initially recognizes the LC molecule on membranes via its ... More
Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy. Less
Angiotensin II receptors AT R and AT R serve as key components of the renin-angiotensin-aldosterone system While AT R plays a central role in the regulation of blood pressure the function of AT R is enigmatic with a variety of reported effects To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors we report crystal structures of the human AT R bound to an AT R-selective and an AT R AT R-dual ligand respectively capturing the receptor in an active-like conformation Unexpectedly helix VIII was found in a non-canonical position stabilizing the active-like state but at ... More
Angiotensin II receptors, AT1R and AT2R, serve as key components of the renin-angiotensin-aldosterone system. While AT1R plays a central role in the regulation of blood pressure, the function of AT2R is enigmatic with a variety of reported effects. To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors, we report crystal structures of the human AT2R bound to an AT2R-selective and an AT1R/AT2R-dual ligand, respectively, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins/β-arrestins, in agreement with the lack of signaling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the interactions critical for ligand binding and selectivity. Our results thus provide insights into the structural basis for distinct functions of the angiotensin receptors, and may guide the design of novel selective ligands. Less
Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids Given the insolubility of the natural C C fatty acyl substrate of the PpsC dehydratase DH domain we investigated its structure function relationships in the presence of shorter surrogate substrates Since most enzymes belonging to the R -specific enoyl hydratase hydroxyacyl dehydratase family conduct the reverse hydration reaction ... More
Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine. The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids. Given the insolubility of the natural C28?C30 fatty acyl substrate of the PpsC dehydratase (DH) domain, we investigated its structure?function relationships in the presence of shorter surrogate substrates. Since most enzymes belonging to the (R)-specific enoyl hydratase/hydroxyacyl dehydratase family conduct the reverse hydration reaction in vitro, we have determined the X-ray structures of the PpsC DH domain, both unliganded (apo) and in complex with trans-but-2-enoyl-CoA or trans-dodec-2-enoyl-CoA derivatives. This study provides for the first time a snapshot of dehydratase?ligand interactions following a hydration reaction. Our structural analysis allowed us to identify residues essential for substrate binding and activity. The structural comparison of the two complexes also sheds light on the need for long acyl chains for this dehydratase to carry out its function, consistent with both its in vitro catalytic behavior and the physiological role of the PpsC enzyme. Less
Insulin-regulated aminopeptidase IRAP is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates although the mechanism behind this wide specificity is not clearly understood We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at resolution In the presence of this inhibitor the enzyme adopts a novel conformation in which domains II and IV are juxtaposed forming a hollow structure that excludes external solvent access to the catalytic center A loop adjacent to the enzyme s GAMEN motif undergoes structural reconfiguration allowing the ... More
Insulin-regulated aminopeptidase (IRAP) is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates, although the mechanism behind this wide specificity is not clearly understood. We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at 2.53 � resolution. In the presence of this inhibitor, the enzyme adopts a novel conformation in which domains II and IV are juxtaposed, forming a hollow structure that excludes external solvent access to the catalytic center. A loop adjacent to the enzyme�s GAMEN motif undergoes structural reconfiguration, allowing the accommodation of bulky inhibitor side chains. Atomic interactions between the inhibitor and IRAP that are unique to this conformation can explain the strong selectivity compared to homologous aminopeptidases ERAP1 and ERAP2. This conformation provides insight on IRAP�s catalytic cycle and reveals significant active-site plasticity that may underlie its substrate permissiveness. Less
This multidisciplinary project begins with one overarching aim to elucidate the role of the rigidity of the lipid tail on the phase transitions of lipidic mesophases Previous studies have demonstrated that the position and the number of cis double bonds in monoacylglycerols determine the chain splay of the molecule establishing how this parameter was essential in influencing the phase behavior Following on from this novel lipids which are inspired by naturally occuring cyclopropanated lipids have been synthesized and their phase behavior elucidated The chain rigidity has been systematically varied by locking the cis configuration of the double bond on the ... More
This multidisciplinary project begins with one overarching aim: to elucidate the role of the
rigidity of the lipid tail on the phase transitions of lipidic mesophases. Previous studies have
demonstrated that the position and the number of cis double bonds in monoacylglycerols
determine the chain splay of the molecule, establishing how this parameter was essential in
influencing the phase behavior. Following on from this, novel lipids which are inspired by
naturally occuring cyclopropanated lipids have been synthesized, and their phase behavior
elucidated. The chain rigidity has been systematically varied by locking the cis configuration
of the double bond on the alkyl chain in a confined geometry. To understand the relationship
between chain rigidity and phase behavior a library of new lipids has been synthesized replacing
the cis double bond by a geometrically confined cyclopropyl ring. The replacement of the
double bond with a chemically analogous cyclopropyl group was designed in order to maintain
a similar chain splay and CPP parameter. The insertion of an additional carbon into the lipidic
chain doesn�t significantly change the length or the curvature of the chain but varies
substantially the packing frustration and the lateral stress of the lipid.
The phase behavior of these novel lipids with identical head group and different alkyl chains
has been investigated with utmost care. Small angle X-ray scattering (SAXS) measurements at
different hydration level and at different temperatures have been used to study the thermal
behavior of these lipid and the effect of this novel motif on the lipidic packing, with particular
attention to low temperature effects.
Since cyclopropanated lipids are present in several dairy products, and since lipidic
nanoparticles have been proved to be excellent drug delivery systems, digestion studies of
cubosomes and hexosomes formed by the novel synthesized cyclopropanated lipids have been
performed. Time resolved synchrotron SAXS has been used to monitor the phase changes
during the enzymatic reaction.
In order to test the utility of the cyclopropanated lipidic systems for membrane protein
crystallization the novel lipidic cubic phase (LCP) matrices have been employed in
crystallization studies with the membrane protein model system bacteriorhodopsin (bR).
IV
Finally, the successful crystallization attempts for membrane protein structural studies of the
chloride channels EcClC and Rm1ClC, as well as the lipopolysaccharide transporter LptD-LptE
show the broad applicability of the LCP crystallization method and the utility of tuning
crystallization conditions, including a screening of different lipids, to optimize crystal growt. Less
rigidity of the lipid tail on the phase transitions of lipidic mesophases. Previous studies have
demonstrated that the position and the number of cis double bonds in monoacylglycerols
determine the chain splay of the molecule, establishing how this parameter was essential in
influencing the phase behavior. Following on from this, novel lipids which are inspired by
naturally occuring cyclopropanated lipids have been synthesized, and their phase behavior
elucidated. The chain rigidity has been systematically varied by locking the cis configuration
of the double bond on the alkyl chain in a confined geometry. To understand the relationship
between chain rigidity and phase behavior a library of new lipids has been synthesized replacing
the cis double bond by a geometrically confined cyclopropyl ring. The replacement of the
double bond with a chemically analogous cyclopropyl group was designed in order to maintain
a similar chain splay and CPP parameter. The insertion of an additional carbon into the lipidic
chain doesn�t significantly change the length or the curvature of the chain but varies
substantially the packing frustration and the lateral stress of the lipid.
The phase behavior of these novel lipids with identical head group and different alkyl chains
has been investigated with utmost care. Small angle X-ray scattering (SAXS) measurements at
different hydration level and at different temperatures have been used to study the thermal
behavior of these lipid and the effect of this novel motif on the lipidic packing, with particular
attention to low temperature effects.
Since cyclopropanated lipids are present in several dairy products, and since lipidic
nanoparticles have been proved to be excellent drug delivery systems, digestion studies of
cubosomes and hexosomes formed by the novel synthesized cyclopropanated lipids have been
performed. Time resolved synchrotron SAXS has been used to monitor the phase changes
during the enzymatic reaction.
In order to test the utility of the cyclopropanated lipidic systems for membrane protein
crystallization the novel lipidic cubic phase (LCP) matrices have been employed in
crystallization studies with the membrane protein model system bacteriorhodopsin (bR).
IV
Finally, the successful crystallization attempts for membrane protein structural studies of the
chloride channels EcClC and Rm1ClC, as well as the lipopolysaccharide transporter LptD-LptE
show the broad applicability of the LCP crystallization method and the utility of tuning
crystallization conditions, including a screening of different lipids, to optimize crystal growt. Less
Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme
During production purification formulation and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability Such harmful conditions include extreme pH changes high ionic strengths or elevated temperatures The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development In this context PEGylation i e the covalent attachment of polyethylene glycol PEG to proteins represents a valuable strategy to improve the physico-chemical properties of proteins In this work the influence ... More
During production, purification, formulation, and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability. Such harmful conditions include extreme pH changes, high ionic strengths or elevated temperatures. The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation, as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development. In this context PEGylation, i.e. the covalent attachment of polyethylene glycol (PEG) to proteins, represents a valuable strategy to improve the physico-chemical properties of proteins. In this work, the influence of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme is investigated. Specifically, conformational and colloidal properties were studied by means of high-throughput melting point determination and automated generation of protein phase diagrams, respectively. Lysozyme from chicken egg-white as a model protein was randomly conjugated to 2 kDa, 5 kDa and 10 kDa mPEG-aldehyde and resulting PEGamer species were purified by chromatographic separation. Besides protein stability assessment, residual enzyme activities were evaluated employing a Micrococcus lysodeikticus based activity assay. PEG molecules with lower molecular weights and lower PEGylation degrees resulted in higher residual activities. Changes in enzyme activities upon PEGylation have shown to result from a combination of steric hindrance and molecular flexibility. In contrast, higher PEG molecular weights and PEGylation degrees enhanced conformational and colloidal stability. By PEGylating lysozyme an increase of the protein solubility by more than 11-fold was achieved. Less
Influenza A haemagglutinin is a surface glycoprotein of Influenza virus responsible for the initial attachment of the virus to the target cell and at a later stage for viral membrane fusion At the acidic pH of the endosome the HA molecule undergoes an irreversible structural rearrangement In consequence the hydrophobic terminal segments of HA are moved to the same end of the refolded molecule promoting membrane fusion haemagglutinin subtypes H -H identified to date can be divided into two groups based on characteristic structural features The low pH-induced structures of proteolytically prepared and E coli-expressed fragments of influenza A H ... More
Influenza A haemagglutinin is a surface glycoprotein of Influenza virus,
responsible for the initial attachment of the virus to the target cell and, at a later
stage, for viral membrane fusion. At the acidic pH of the endosome, the HA
molecule undergoes an irreversible structural rearrangement. In consequence, the
hydrophobic terminal segments of HA2 are moved to the same end of the refolded
molecule, promoting membrane fusion.
16 haemagglutinin subtypes (H1-H16) identified to date can be divided into two
groups based on characteristic structural features. The low pH-induced structures
of proteolytically prepared and E.coli-expressed fragments of influenza A H3 HA2
(group 2 HA) were previously determined by X-ray crystallography.
This study presents structures of proteolytically prepared and recombinantlyexpressed fragments of H1 HA2 in a postfusion conformation. Refolded H1 HA2,
belonging to group 1 HA, adopts a hairpin-like conformation, similar to that of a
rearranged H3 HA2. Structures were compared to the known structures of low pHactivated HA2, to gain a better understanding of the structural differences between
the two groups of HA.
The data show the structures of the refolded HA2 to be conserved between the HA
groups with minor differences.
These structural data are supplemented with functional studies involving the
cross-reactive FI6 antibody. FI6 antibody binds near the conserved fusion
subdomain of the HA molecule and thus interferes with the low pH-triggered
conformational change of HA. Additional methods employed in this study, such as
limited proteolysis, electron microscopy, biolayer interferometry and MDCK1 cell
infection, give insight into the mechanism of FI6 antibody-mediated neutralization,
and highlight the differences in infectivity of H1N1 and H3N2 viruses neutralized
by the FI6 antibody. Less
responsible for the initial attachment of the virus to the target cell and, at a later
stage, for viral membrane fusion. At the acidic pH of the endosome, the HA
molecule undergoes an irreversible structural rearrangement. In consequence, the
hydrophobic terminal segments of HA2 are moved to the same end of the refolded
molecule, promoting membrane fusion.
16 haemagglutinin subtypes (H1-H16) identified to date can be divided into two
groups based on characteristic structural features. The low pH-induced structures
of proteolytically prepared and E.coli-expressed fragments of influenza A H3 HA2
(group 2 HA) were previously determined by X-ray crystallography.
This study presents structures of proteolytically prepared and recombinantlyexpressed fragments of H1 HA2 in a postfusion conformation. Refolded H1 HA2,
belonging to group 1 HA, adopts a hairpin-like conformation, similar to that of a
rearranged H3 HA2. Structures were compared to the known structures of low pHactivated HA2, to gain a better understanding of the structural differences between
the two groups of HA.
The data show the structures of the refolded HA2 to be conserved between the HA
groups with minor differences.
These structural data are supplemented with functional studies involving the
cross-reactive FI6 antibody. FI6 antibody binds near the conserved fusion
subdomain of the HA molecule and thus interferes with the low pH-triggered
conformational change of HA. Additional methods employed in this study, such as
limited proteolysis, electron microscopy, biolayer interferometry and MDCK1 cell
infection, give insight into the mechanism of FI6 antibody-mediated neutralization,
and highlight the differences in infectivity of H1N1 and H3N2 viruses neutralized
by the FI6 antibody. Less
Eps epidermal growth factor receptor pathway substrate -homology domain containing proteins EHDs are molecular machines that use the energy of ATP binding and ATP hydrolysis to remodel shallow membranes into highly curved membrane tubules This activity is required in many cellular membrane trafficking pathways In this work we have determined a high-resolution structure of an EHD machine in the active state The structure indicates how EHDs assemble at the membrane surface into ring-like scaffolds that deform the underlying membrane By comparing this active state with a previously determined autoinhibited conformation we can deduce the mechanistic details how recruitment of EHDs ... More
Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) are molecular machines that use the energy of ATP binding and ATP hydrolysis to remodel shallow membranes into highly curved membrane tubules. This activity is required in many cellular membrane trafficking pathways. In this work, we have determined a high-resolution structure of an EHD machine in the active state. The structure indicates how EHDs assemble at the membrane surface into ring-like scaffolds that deform the underlying membrane. By comparing this active state with a previously determined autoinhibited conformation, we can deduce the mechanistic details how recruitment of EHDs to membranes is regulated. A comparison with other membrane-associated molecular machines reveals commonalities and differences in the activation mechanism. Less
Eps epidermal growth factor receptor pathway substrate -homology domain containing proteins EHDs comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking EHD proteins consist of a dynamin-related GTPase domain a helical domain and a C-terminal Eps -homology EH domain Previous studies have revealed the structure of the EHD dimer Furthermore the N terminal region of EHD was demonstrated to bind to a hydrophobic groove of the GTPase domain and to switch into the membrane in the presence of liposome suggesting an autoinhibitory role However the molecular mechanisms of membrane binding oligomerization and nucleotide hydrolysis have remained obscure ... More
Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. EHD proteins consist of a dynamin-related GTPase domain, a helical domain and a C-terminal Eps15-homology (EH) domain,. Previous studies have revealed the structure of the EHD2 dimer. Furthermore, the N terminal region of EHD2 was demonstrated to bind to a hydrophobic groove of the GTPase domain and to switch into the membrane in the presence of liposome, suggesting an autoinhibitory role However, the molecular mechanisms of membrane binding, oligomerization and nucleotide hydrolysis have remained obscure. To understand the mechanism of membrane recruitment, the crystal structure of an aminoterminally truncated EHD4 dimer in complex with ATPγS and ADP were determined in this thesis. Compared with the EHD2 structure, the helical domains assume an open conformation featuring a 50° rotation relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), it was shown that the opening aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop region in the GTPase domain undergoes a large rearrangement and creates a new interface that allows oligomerization on membranes. These results suggest that the EHD4 structures represent the active EHD conformation, whereas the EHD2 structure is autoinhibited. A model for the activation and oligomerization of EHD proteins was proposed in which a series of domain rearrangements control membrane recruitment and remodeling in the EHD family. A comparison with other peripheral membrane proteins elucidated common and specific features of this activation mechanism. Less
Type phosphatidic acid phosphatases PAP s can be either soluble or integral membrane enzymes In bacteria integral membrane PAP s play major roles in the metabolisms of glycerophospholipids undecaprenyl-phosphate C -P lipid carrier and lipopolysaccharides By in vivo functional experiments and biochemical characterization we show that the membrane PAP coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate PGP phosphatase of B subtilis We also confirm that this enzyme renamed bsPgpB has a weaker activity on C -PP Moreover we solved the crystal structure of bsPgpB at resolution with tungstate a phosphate analog in the active site ... More
Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s. Less
The Haemophilus surface fibril Hsf is an unusually large trimeric autotransporter adhesin TAA expressed by the most virulent strains of H influenzae Hsf is known to mediate adhesion between pathogen and host allowing the establishment of potentially deadly diseases such as epiglottitis meningitis and pneumonia While recent research has suggested that this TAA might adopt a novel hairpin-like architecture the characterization of Hsf has been limited to in silico modelling and electron micrographs with no high-resolution structural data available Here the crystal structure of Hsf putative domain PD is reported at resolution The structure corrects the previous domain annotation by ... More
The Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel ‘hairpin-like’ architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the ‘hairpin-like’ hypothesis. Less
Current influenza vaccines are mostly formulated as liquids which requires a continuous cold chain to maintain the stability of the antigen For development of vaccines with an increased stability at ambient temperatures manifold parameters and their influences on the colloidal stability and activity of the antigen have to be understood This work presents a strategy to examine both the colloidal stability and the remaining biological activity of H N influenza viruses under various conditions after an incubation of days H N phase diagrams were generated for several pH values and different initial H N and NaCl concentrations It was shown ... More
Current influenza vaccines are mostly formulated as liquids which requires a continuous cold chain to maintain the stability of the antigen. For development of vaccines with an increased stability at ambient temperatures, manifold parameters and their influences on the colloidal stability and activity of the antigen have to be understood. This work presents a strategy to examine both, the colloidal stability and the remaining biological activity of H1N1 influenza viruses under various conditions after an incubation of 40 days. H1N1 phase diagrams were generated for several pH values and different initial H1N1 and NaCl concentrations. It was shown that the highest H1N1 recoveries were obtained for pH 6 and that moderate amounts of NaCl are favorable for increased recoveries. In contrast to colloidal stability, the highest remaining HA activity was observed at pH 9. The electrostatic and hydrophobic surface properties of H1N1 were investigated to reveal the mechanisms accounting for the decrease in stability. Secondly, the capability of virus precipitation by polyethylene glycol in combination with determination of surface hydrophobicity was proven to be useful as a predictive tool to rank stability under different conditions. This methodology enables the rapid assessment of aggregation propensity of H1N1 formulations and the influence on the activity of the virus particles and might become a standard tool during the development of vaccine formulations. Less
Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals Interestingly phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages Here we demonstrate that peroxidase A from D discoideum DdPoxA is a stable monomeric glycosylated and secreted heme peroxidase with homology to mammalian peroxidases The first crystal structure resolution of a mycetozoan peroxidase of this superfamily shows ... More
Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals. Interestingly, phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum. This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages. Here, we demonstrate that peroxidase A from D. discoideum (DdPoxA) is a stable, monomeric, glycosylated, and secreted heme peroxidase with homology to mammalian peroxidases. The first crystal structure (2.5 Å resolution) of a mycetozoan peroxidase of this superfamily shows the presence of a post-translationally-modified heme with one single covalent ester bond between the 1-methyl heme substituent and Glu-236. The metalloprotein follows the halogenation cycle, whereby compound I oxidizes iodide and thiocyanate at high rates (>108 m−1 s−1) and bromide at very low rates. It is demonstrated that DdPoxA is up-regulated and likely secreted at late multicellular development stages of D. discoideum when migrating slugs differentiate into fruiting bodies that contain persistent spores on top of a cellular stalk. Expression of DdPoxA is shown to restrict bacterial contamination of fruiting bodies. Structure and function of DdPoxA are compared with evolutionary-related mammalian peroxidases in the context of non-specific immune defense. Less
Membrane-like nanodiscs ND have become an important tool for the cell-free expression solubilization folding and in vitro structural and functional studies of membrane proteins MPs Direct crystallization of MPs embedded in NDs would be of high importance for structural biology However despite considerable efforts we have been as yet unable to obtain crystals suitable for X-ray crystallography In the present work we show that an ND-trapped MP can be transferred into the cubic phase and crystallized in meso Bacteriorhodopsin BR reconstituted into nanodiscs was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant The resulting crystals ... More
Membrane-like nanodiscs (ND) have become an important tool for the cell-free expression, solubilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs embedded in NDs would be of high importance for structural biology. However, despite considerable efforts we have been as yet unable to obtain crystals suitable for X-ray crystallography. In the present work, we show that an ND-trapped MP can be transferred into the cubic phase and crystallized in meso. Bacteriorhodopsin (BR) reconstituted into nanodiscs was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant. The resulting crystals diffract beyond 1.8 Å. The structure of BR was solved at 1.9 Å and found to be indistinguishable from previous structures obtained with the protein solubilized in detergent. We suggest the proposed protocol of in meso crystallization to be generally applicable to ND-trapped MPs. Less
Riboswitches are structural RNA elements that are generally located in the ' untranslated region of messenger RNA During regulation of gene expression ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time Here we use femtosecond X-ray free electron laser XFEL pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction We demonstrate this approach by determining four structures of the ... More
Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform1�3. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time4. Here we use femtosecond X-ray free electron laser (XFEL) pulses5,6 to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of �mix-and-inject� time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes. Less
By interacting with hundreds of protein partners - - proteins coordinate vital cellular processes Phosphorylation of the small heat shock protein HSPB within its intrinsically disordered N-terminal domain activates its interaction with - - ultimately triggering smooth muscle relaxation After analyzing the binding of an HSPB -derived phosphopeptide to - - using isothermal calorimetry and X-ray crystallography we have determined the crystal structure of the complete assembly consisting of the - - dimer and full-length HSPB dimer and further characterized this complex in solution using fluorescence spectroscopy small-angle X-ray scattering and limited proteolysis We show that selected intrinsically disordered regions ... More
By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein HSPB6 within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first-ever atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants. Less
Studies of the intermediate filament IF structure are a prerequisite of understanding their function In addition the structural information is indispensable if one wishes to gain a mechanistic view on the disease-related mutations in the IFs Over the years considerable progress has been made on the atomic structure of the elementary building block of all IFs the coiled-coil dimer Here we discuss the approaches methods and practices that have contributed to this advance With abundant genetic information on hand bioinformatics approaches give important insights into the dimer structure including the head and tail regions poorly assessable experimentally At the same ... More
Studies of the intermediate filament (IF) structure are a prerequisite of understanding their function. In addition, the structural information is indispensable if one wishes to gain a mechanistic view on the disease-related mutations in the IFs. Over the years, considerable progress has been made on the atomic structure of the elementary building block of all IFs, the coiled-coil dimer. Here, we discuss the approaches, methods and practices that have contributed to this advance. With abundant genetic information on hand, bioinformatics approaches give important insights into the dimer structure, including the head and tail regions poorly assessable experimentally. At the same time, the most important contribution has been provided by X-ray crystallography. Following the “divide-and-conquer” approach, many fragments from several IF proteins could be crystallized and resolved to atomic resolution. We will systematically cover the main procedures of these crystallographic studies, suggest ways to maximize their efficiency, and also discuss the possible pitfalls and limitations. In addition, electron paramagnetic resonance with site-directed spin labeling was another method providing a major impact toward the understanding of the IF structure. Upon placing the spin labels into specific positions within the full-length protein, one can evaluate the proximity of the labels and their mobility. This makes it possible to make conclusions about the dimer structure in the coiled-coil region and beyond, as well as to explore the dimer–dimer contacts. Less
The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand With the upstream improvements the bottleneck shifted towards the downstream process New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules overcoming the current chromatographic bottlenecks Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules This review focuses in the role of these two methodologies in current ... More
The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups. Less
In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity To guarantee stable biopharmaceutical processes and safe formulations both phenomenons have to be avoided as these may hinder regular processing steps This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions For this purpose additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected These additives were PEG PEG glycerol glycine NaCl and ArgHCl Concentrated lysozyme and glucose oxidase solutions at pH and served as ... More
In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity. To guarantee stable biopharmaceutical processes and safe formulations, both phenomenons have to be avoided as these may hinder regular processing steps. This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions. For this purpose, additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected. These additives were PEG 300, PEG 1000, glycerol, glycine, NaCl and ArgHCl. Concentrated lysozyme and glucose oxidase solutions at pH 3 and 9 served as model systems. Fourier-transformed-infrared spectroscopy was chosen to determine the conformational stability of selected protein samples. Influencing protein interactions, the impact of additives was strongly dependent on pH. Of all additives investigated, glycine was the only one that maintained protein conformational and colloidal stability while decreasing the dynamic viscosity. Low concentrations of NaCl showed the same effect, but increasing concentrations resulted in visible protein aggregates. Less
PRMT is the less-characterized member of the protein arginine methyltransferase family in terms of structure activity and cellular functions PRMT is a modular protein containing a catalytic Ado-Met-binding domain and unique Src homology domain that binds proteins with proline-rich motifs PRMT is involved in a variety of cellular processes and has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners PRMT has been demonstrated to have weak methyltransferase activity on a histone H substrate but its optimal substrates have not yet been identified To obtain insights into the function and activity of PRMT we solve several ... More
PRMT2 is the less-characterized member of the protein arginine methyltransferase family in terms of structure, activity, and cellular functions. PRMT2 is a modular protein containing a catalytic Ado-Met-binding domain and unique Src homology 3 domain that binds proteins with proline-rich motifs. PRMT2 is involved in a variety of cellular processes and has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners. PRMT2 has been demonstrated to have weak methyltransferase activity on a histone H4 substrate, but its optimal substrates have not yet been identified. To obtain insights into the function and activity of PRMT2, we solve several crystal structures of PRMT2 from two homologs (zebrafish and mouse) in complex with either the methylation product S-adenosyl-L-homocysteine or other compounds including the first synthetic PRMT2 inhibitor (Cp1) studied so far. We reveal that the N-terminal-containing SH3 module is disordered in the full-length crystal structures, and highlights idiosyncratic features of the PRMT2 active site. We identify a new nonhistone protein substrate belonging to the serine-/arginine-rich protein family which interacts with PRMT2 and we characterize six methylation sites by mass spectrometry. To better understand structural basis for Cp1 binding, we also solve the structure of the complex PRMT4:Cp1. We compare the inhibitor–protein interactions occurring in the PRMT2 and PRMT4 complex crystal structures and show that this compound inhibits efficiently PRMT2. These results are a first step toward a better understanding of PRMT2 substrate recognition and may accelerate the development of structure-based drug design of PRMT2 inhibitors. Less
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators Here we report the generation of a quantitative interaction network directly linking human proteins to the AAA ATPase p an essential hexameric protein with multiple cellular functions We show that the high-affinity interacting protein ASPL efficiently promotes p hexamer disassembly resulting in the formation of stable p ASPL heterotetramers High-resolution structural and biochemical studies indicate that an extended UBX domain eUBX in ASPL is critical for p hexamer disassembly and facilitates the assembly of p ASPL heterotetramers This spontaneous process is accompanied by ... More
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity. Less
Cannabinoid receptor CB is the principal target of -tetrahydrocannabinol THC a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use CB is activated by endocannabinoids and is a promising therapeutic target for pain management inflammation obesity and substance abuse disorders Here we present the crystal structure of human CB in complex with AM a stabilizing antagonist synthesized and characterized for this structural study The structure of the CB -AM complex reveals key features of the receptor and critical interactions for antagonist binding In combination with functional studies and molecular modeling ... More
Cannabinoid receptor 1 (CB1) is the principal target of ?9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 � crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals. Less
Tn and Tn -like transposons are complex elements found in disparate environments and are responsible for mobilizing a wide variety of genes and forming pathogenicity fitness islands They are novel in their ability to recognize both a single site in the chromosome and specifically target transposition into mobile plasmids via dedicated TnsD and TnsE targeting proteins TnsE recognizes mobile plasmids through an association with the processivity clamp and a ' recessed DNA end during conjugal replication However the mechanism for the specific recognition of ' recessed DNA ends remains unclear Structural analyses of the C-terminal domain of TnsE identified a ... More
Tn7 and Tn7-like transposons are complex elements found in disparate environments and are responsible for mobilizing a wide variety of genes and forming pathogenicity/fitness islands. They are novel in their ability to recognize both a single site in the chromosome and specifically target transposition into mobile plasmids via dedicated TnsD and TnsE targeting proteins. TnsE recognizes mobile plasmids through an association with the processivity clamp and a 3' recessed DNA end during conjugal replication. However, the mechanism for the specific recognition of 3' recessed DNA ends remains unclear. Structural analyses of the C-terminal domain of TnsE identified a novel protein fold
including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-function variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structurebased analysis of a series of TnsE variants relates transposition to DNA binding stability. Follow up studies of full length TnsE bound to DNA are in progress. Less
including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-function variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structurebased analysis of a series of TnsE variants relates transposition to DNA binding stability. Follow up studies of full length TnsE bound to DNA are in progress. Less
Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase CutC has emerged as an important route for the production of trimethylamine a microbial metabolite associated with both human disease and biological methane production Here we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline Within an unexpectedly polar active site CutC orients choline through hydrogen bonding with a putative general base and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety These structural data along with ... More
Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as an important route for the production of trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structural data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of trimethylamine. This work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production. Less
The colloidal stability of a protein solution during downstream processing formulation and storage is a key issue for the biopharmaceutical production process Thus knowledge about colloidal solution characteristics such as the tendency to form aggregates or high viscosity at various processing conditions is of interest This work correlates changes in the apparent diffusion coefficient as a parameter of protein interactions with observed protein aggregation and dynamic viscosity of the respective protein samples For this purpose the diffusion coefficient the protein phase behavior and the dynamic viscosity in various systems containing the model proteins -lactalbumin lysozyme and glucose oxidase were studied ... More
The colloidal stability of a protein solution during downstream processing, formulation, and storage is a key issue for the biopharmaceutical production process. Thus, knowledge about colloidal solution characteristics, such as the tendency to form aggregates or high viscosity, at various processing conditions is of interest. This work correlates changes in the apparent diffusion coefficient as a parameter of protein interactions with observed protein aggregation and dynamic viscosity of the respective protein samples. For this purpose, the diffusion coefficient, the protein phase behavior, and the dynamic viscosity in various systems containing the model proteins α-lactalbumin, lysozyme, and glucose oxidase were studied. Each of these experiments revealed a wide range of variations in protein interactions depending on protein type, protein concentration, pH, and the NaCl concentration. All these variations showed to be mirrored by changes in the apparent diffusion coefficient in the respective samples. Whereas stable samples with relatively low viscosity showed an almost linear dependence, the deviation from the concentration-dependent linearity indicated both an increase in the sample viscosity and probability of protein aggregation. This deviation of the apparent diffusion coefficient from concentration-dependent linearity was independent of protein type and solution properties for this study. Thus, this single parameter shows the potential to act as a prognostic tool for colloidal stability of protein solutions. Less
Human metapneumovirus hMPV is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age The hMPV fusion F glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen To facilitate structure-based vaccine design we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of by X-ray crystallography The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus hRSV In contrast significant differences were apparent ... More
Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses. Less
In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step Unwanted protein precipitation or crystallization can lead to column pipe or filter blocking In formulation the formation of aggregates can even be lethal when injected into the patient The typical methodology to illustrate protein phase states is the generation of protein phase diagrams Commonly protein phase behavior is shown in dependence of protein and precipitant concentration Despite using high-throughput methods for the generation of phase diagrams the time necessary to reach equilibrium is the bottleneck ... More
In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step. Unwanted protein precipitation or crystallization can lead to column, pipe or filter blocking. In formulation, the formation of aggregates can even be lethal when injected into the patient. The typical methodology to illustrate protein phase states is the generation of protein phase diagrams. Commonly, protein phase behavior is shown in dependence of protein and precipitant concentration. Despite using high-throughput methods for the generation of phase diagrams, the time necessary to reach equilibrium is the bottleneck. Faster methods to predict protein phase behavior are desirable. In this study, hydrophobic interaction chromatography retention times were correlated to crystal size and form. High-throughput thermal stability measurements (melting and aggregation temperatures), using an Optim®2 system, were successfully correlated to glucose isomerase stability. By using hydrophobic interaction chromatography and thermal stability determinations, glucose isomerase conformational and colloidal stability were successfully predicted for different salts in a specific pH range. Less
Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation However alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease Glycogen phosphorylase GP a key enzyme in glycogen metabolism catalyzes the rate-limiting step of glycogen mobilization Moreover the allosteric regulation of the three GP isozymes muscle liver and brain by metabolites and phosphorylation in response to hormonal signaling fine-tunes glycogenolysis to fulfill energetic and metabolic requirements Whereas the structures of muscle and liver GPs have been known for decades the structure of brain GP ... More
Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 �) and in complex with its allosteric activator AMP (3.4 �). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen. Less
Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures and most VacA activities are dependent on its capacity to oligomerize The -kDa secreted VacA protein can undergo limited proteolysis to yield two domains designated p and p The p domain is required for membrane channel formation and intracellular toxic activities and the p domain has an important role in mediating VacA binding to cells Previous studies showed that the p domain has a predominantly -helical structure ... More
Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly �-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the �-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the �-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly. Less
Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM Here we present the crystal structure of the C perfringens S SleM protein at SleM comprises an N-terminal catalytic domain that adopts an irregular -barrel fold that is common to GH family lysozymes plus a C-terminal fibronectin type III domain The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution A truncated form of SleM that lacks the FnIII domain ... More
Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination. SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM. Here, we present the crystal structure of the�C. perfringens�S40 SleM protein at 1.8 �. SleM comprises an N-terminal catalytic domain that adopts an irregular ?/?-barrel fold that is common to GH25 family lysozymes, plus a C-terminal fibronectin type III domain. The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution. A truncated form of SleM that lacks the FnIII domain shows reduced activity against spore sacculi indicating that this domain may have a role in facilitating the position of substrate with respect to the enzyme's active site. Less
Nonlinear optical methods such as second harmonic generation SHG and two-photon excited UV fluorescence TPE-UVF imaging are promising approaches to address bottlenecks in the membrane protein structure determination pipeline The general principles of SHG and TPE-UVF are discussed here along with instrument design considerations Comparisons to conventional methods in high throughput crystallization condition screening and crystal quality assessment prior to X-ray diffraction are also discussed
Chromosome integrity depends on DNA structure-specific processing complexes that resolve DNA entanglement between sister chromatids If left unresolved these entanglements can generate either chromatin bridging or ultrafine DNA bridging in the anaphase of mitosis These bridge structures are defined by the presence of the PICH protein which interacts with the BEND protein in mitosis To obtain structural insights into PICH BEND complex formation at the atomic level their respective NTPR and BD domains were cloned overexpressed and crystallized using M ammonium sulfate as a precipitant at pH The protein complex readily formed large hexagonal crystals belonging to space group P ... More
Chromosome integrity depends on DNA structure-specific processing complexes that resolve DNA entanglement between sister chromatids. If left unresolved, these entanglements can generate either chromatin bridging or ultrafine DNA bridging in the anaphase of mitosis. These bridge structures are defined by the presence of the PICH protein, which interacts with the BEND3 protein in mitosis. To obtain structural insights into PICH�BEND3 complex formation at the atomic level, their respective NTPR and BD1 domains were cloned, overexpressed and crystallized using 1.56 M ammonium sulfate as a precipitant at pH 7.0. The protein complex readily formed large hexagonal crystals belonging to space group P6122, with unit-cell parameters a = b = 47.28, c = 431.58 � and with one heterodimer in the asymmetric unit. A complete multiwavelength anomalous dispersion (MAD) data set extending to 2.2 � resolution was collected from a selenomethionine-labelled crystal at the Swiss Light Source. Less
A new approach is described to screen for protein nanocrystals based on the reversibility of crystallization Methods to characterize nanocrystals are in strong need to facilitate sample preparation for serial femtosecond X-ray nanocrystallography SFX SFX enables protein structure determination by collecting X-ray diffraction from nano- and microcrystals using a free electron laser This technique is especially valuable for challenging proteins as for example membrane proteins and is in general a powerful method to overcome the radiation damage problem and to perform time-resolved structure analysis Nanocrystal growth cannot be monitored with common methods used in protein crystallography as the resolution of ... More
A new approach is described to screen for protein nanocrystals based on the reversibility of crystallization. Methods to characterize nanocrystals are in strong need to facilitate sample preparation for serial femtosecond X-ray nanocrystallography (SFX). SFX enables protein structure determination by collecting X-ray diffraction from nano- and microcrystals using a free electron laser. This technique is especially valuable for challenging proteins as for example membrane proteins and is in general a powerful method to overcome the radiation damage problem and to perform time-resolved structure analysis. Nanocrystal growth cannot be monitored with common methods used in protein crystallography, as the resolution of bright field microscopy is not sufficient. A high-performance method to screen for nanocrystals is second order nonlinear imaging of chiral crystals (SONICC). However, the high cost prevents its use in every laboratory, and some protein nanocrystals may be �invisible� to SONICC. In this work using a crystallization robot and a common imaging system precipitation comprised of nanocrystals and precipitation caused by aggregated protein can be distinguished. Less
This is a series of investigations into the molecular basis of the evolution of new protein functions The broad objective of this work was to determine exactly how a series of single amino acid mutations typical of an evolutionary trajectory can result in dramatic changes in catalytic activity specificity and protein solubility Various strategies were employed to achieve this aim including analysis of existing literature concerning the various models and theories relating to molecular evolution protein crystallography extensive enzyme kinetics and thermodynamic analysis theoretical analysis of catalytic mechanisms and computational simulation of protein dynamics Three model systems were investigated the ... More
This is a series of investigations into the molecular basis of the evolution of new protein functions. The broad objective of this work was to determine exactly how a series of single amino acid mutations, typical of an evolutionary trajectory, can result in dramatic changes in catalytic activity, specificity and protein solubility. Various strategies were employed to achieve this aim, including analysis of existing literature concerning the various models and theories relating to molecular evolution, protein crystallography, extensive enzyme kinetics and thermodynamic analysis, theoretical analysis of catalytic mechanisms and computational simulation of protein dynamics. Three model systems were investigated: the de novo designed Kemp Eliminase (KE07), the metallo-β-lactamases NDM1 and VIM2, and the N-acylhomoserine lactonase AiiA. Based on these studies, I was able to identify three clear phenomena that are important in molecular evolution: first, preorganization of the active sites residues is essential for efficient catalysis; second, remote mutations are capable of causing quite drastic rearrangements to the active site and substrate binding site by modulating the conformational landscape of a protein; third, intramolecular epistasis, the way that mutations interact with each other and the sequence background that they are introduced to, can constrain evolutionary trajectories and make the evolutionary potential of a protein contingent on its starting sequence. In Chapters 3-5 I focus on KE07, performing detailed kinetic analysis of hydrogenated and deuterated substrate, which revealed entropy-enthalpy compensation in the improvement in activity as well as an unusual change in the kinetic properties in the middle of the evolutionary trajectory. This is followed by comprehensive structural analysis, which reveals the enzyme has evolved to adopt a completely unexpected active site configuration via remote mutations. Finally, using computational simulations and solution fluorescence spectroscopy, I confirm that the in crystallo and kinetic observations are consistent with the behaviour of the protein in solution. Chapter 6 consists of a manuscript that describes the effects of conformational tinkering on the N-acyl-homoserine lactonase AiiA, specifically how remote mutations can have dramatic effects on activity by modulating the conformation of the active site. My contribution to this work included crystal structures and molecular dynamics simulations. Finally, Chapter 7 is a second manuscript that focuses on evolutionary contingency: by examining two related subfamilies of the metallo-β-lactamases, NDM1 and VIM2 we show that the evolvability of each is constrained by intramolecular epistasis and contingent on the starting sequence. To achieve the same final goal (greater whole cell activity), NDM1 evolved higher activity, while VIM2 evolved greater solubility. The crystals structures that I solved revealed the structural basis for the enhanced activity in NDM1 and that enhanced solubility in VIM2 is a result of an unprecedented (for an enzyme) structural rearrangement where the two halves of the α/β sandwich metallo- β-lactamase protein fold have separated and rearranged in an domain-swapped dimer. Less
Extraordinary antibodies capable of near pan-neutralization of HIV- have been identified One of the broadest is antibody E which recognizes the membrane-proximal external region MPER of the HIV- envelope and neutralizes of circulating HIV- strains If delivered passively E might serve to prevent or treat HIV- infection Antibody E however is markedly less soluble than other antibodies Here we describe the use of both structural biology and somatic variation to develop optimized versions of E with increased solubility From the structure of E we identified a prominent hydrophobic patch reversion of four hydrophobic residues in this patch to their hydrophilic ... More
Extraordinary antibodies capable of near pan-neutralization of HIV-1 have been identified. One of the broadest is antibody 10E8, which recognizes the membrane-proximal external region (MPER) of the HIV-1 envelope and neutralizes >95% of circulating HIV-1 strains. If delivered passively, 10E8 might serve to prevent or treat HIV-1 infection. Antibody 10E8, however, is markedly less soluble than other antibodies. Here, we describe the use of both structural biology and somatic variation to develop optimized versions of 10E8 with increased solubility. From the structure of 10E8, we identified a prominent hydrophobic patch; reversion of four hydrophobic residues in this patch to their hydrophilic germ line counterparts resulted in an ~10-fold decrease in turbidity. We also used somatic variants of 10E8, identified previously by next-generation sequencing, to optimize heavy and light chains; this process yielded several improved variants. Of these, variant 10E8v4 with 26 changes versus the parent 10E8 was the most soluble, with a paratope we showed crystallographically to be virtually identical to that of 10E8, a potency on a panel of 200 HIV-1 isolates also similar to that of 10E8, and a half-life in rhesus macaques of ~10 days. An anomaly in 10E8v4 size exclusion chromatography that appeared to be related to conformational isomerization was resolved by engineering an interchain disulfide. Thus, by combining a structure-based approach with natural variation in potency and solubility from the 10E8 lineage, we successfully created variants of 10E8 which retained the potency and extraordinary neutralization breadth of the parent 10E8 but with substantially increased solubility. Less
A detailed understanding of chemical and biological function and the mechanisms underlying the molecular activities ultimately requires atomic-resolution structural data Diffraction-based techniques such as single-crystal X-ray crystallography electron microscopy and neutron diffraction are well established and they have paved the road to the stunning successes of modern-day structural biology The major advances achieved in the last years in all aspects of structural research including sample preparation crystallization the construction of synchrotron and spallation sources phasing approaches and high-speed computing and visualization now provide specialists and nonspecialists alike with a steady flow of molecular images of unprecedented detail The present unit ... More
A detailed understanding of chemical and biological function and the mechanisms underlying the molecular activities ultimately requires atomic-resolution structural data. Diffraction-based techniques such as single-crystal X-ray crystallography, electron microscopy, and neutron diffraction are well established and they have paved the road to the stunning successes of modern-day structural biology. The major advances achieved in the last 20 years in all aspects of structural research, including sample preparation, crystallization, the construction of synchrotron and spallation sources, phasing approaches, and high-speed computing and visualization, now provide specialists and nonspecialists alike with a steady flow of molecular images of unprecedented detail. The present unit combines a general overview of diffraction methods with a detailed description of the process of a single-crystal X-ray structure determination experiment, from chemical synthesis or expression to phasing and refinement, analysis, and quality control. For novices it may serve as a stepping-stone to more in-depth treatises of the individual topics. Readers relying on structural information for interpreting functional data may find it a useful consumer guide. Less
Magnetotactic bacteria are Gram-negative bacteria that navigate along geomagnetic fields using the magnetosome an organelle that consists of a membrane-enveloped magnetic nanoparticle Magnetite formation and its properties are controlled by a specific set of proteins MamC is a small magnetosome-membrane protein that is known to be active in iron biomineralization but its mechanism has yet to be clarified Here we studied the relationship between the MamC magnetite-interaction loop MIL structure and its magnetite interaction using an inert biomineralization protein-MamC chimera Our determined structure shows an alpha-helical fold for MamC-MIL with highly charged surfaces Additionally the MamC-MIL induces the formation of ... More
Magnetotactic bacteria are Gram-negative bacteria that navigate along geomagnetic fields using the magnetosome, an organelle that consists of a membrane-enveloped magnetic nanoparticle. Magnetite formation and its properties are controlled by a specific set of proteins. MamC is a small magnetosome-membrane protein that is known to be active in iron biomineralization but its mechanism has yet to be clarified. Here, we studied the relationship between the MamC magnetite-interaction loop (MIL) structure and its magnetite interaction using an inert biomineralization protein-MamC chimera. Our determined structure shows an alpha-helical fold for MamC-MIL with highly charged surfaces. Additionally, the MamC-MIL induces the formation of larger magnetite crystals compared to protein-free and inert biomineralization protein control experiments. We suggest that the connection between the MamC-MIL structure and the protein’s charged surfaces is crucial for magnetite binding and thus for the size control of the magnetite nanoparticles. Less
Haemophilus influenzae is an obligate human commensal pathogen that requires haem for survival and can acquire it from several host haemoproteins including haemopexin The haem transport system from haem-haemopexin consists of HxuC a haem receptor and the two-partner-secretion system HxuB HxuA HxuA which is exposed at the cell surface is strictly required for haem acquisition from haemopexin HxuA forms complexes with haem-haemopexin leading to haem release and its capture by HxuC The key question is how HxuA liberates haem from haemopexin Here we solve crystal structures of HxuA alone and HxuA in complex with the N-terminal domain of haemopexin A ... More
Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin. Less
The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine precision medicine Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules As a consequence individual and tailored productions require generic approaches for a fast and dedicated purification process development In this article different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and ... More
The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine (precision medicine). Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules. As a consequence, individual and tailored productions require generic approaches for a fast and dedicated purification process development. In this article, different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies. Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and mechanistic modeling tools. Such approaches constitute a good foundation for fast and effective process development for new products and processes, but their full potential becomes obvious in a correlated combination. Thus, different combinatorial approaches are presented, which might become future directions in the biopharmaceutical industry. Less
LUX ARRHYTHMO LUX is a Myb-domain transcription factor that plays an important role in regulating the circadian clock Lux mutations cause severe clock defects and arrhythmia in constant light and dark In order to examine the molecular mechanisms underlying the function of LUX the DNA-binding Myb domain was cloned expressed and purified The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays EMSAs demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity In order to investigate the specificity determinants of protein DNA interactions the protein was co-crystallized with a -mer ... More
LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock. Lux mutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein�DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using the MeshAndCollect workflow available at the ESRF. Less
A-kinase anchoring proteins AKAPs interact with the dimerization docking D D domains of regulatory subunits of the ubiquitous protein kinase A PKA AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling Here we elucidated the structure of an extended PKA-binding domain of AKAP bound to the D D domain of the regulatory RIIa subunits of PKA We identified three hydrophilic anchor points in AKAP outside the core PKA-binding domain which mediate contacts with the D D domain Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA We ... More
A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18� bound to the D/D domain of the regulatory RIIa subunits of PKA. We identified three hydrophilic anchor points in AKAP18� outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions. Less
The SCP2-thiolase-like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism
Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP -thiolase-like protein SLP which is characterized by having the YDCF thiolase sequence fingerprint of the C -C loop SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals including human Deletion of the Trypanosoma brucei SLP gene TbSLP increases the doubling time of procyclic T brucei and causes a -fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion The crystal structure ... More
Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2-thiolase-like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2-Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5-fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA. Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP-acetoacetyl-CoA (1.90 Å) and TbSLP-malonyl-CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl-CoA tightly (Kd 90 µM), acetoacetyl-CoA moderately (Kd 0.9 mM) and acetyl-CoA and CoA very weakly. TbSLP possesses low malonyl-CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Less
Serial femtosecond X-ray crystallography SFX using an X-ray free electron laser XFEL is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins including G-protein coupled receptors GPCRs which often only produce microcrystals An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in Here we report the deposition of the XFEL data and provide further details on crystallization XFEL data collection and analysis structure determination and the validation of the structural model The rhodopsin-arrestin crystal structure solved with ... More
Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. Less