Faille, Alexandre, Gavalda, Sabine Insights into substrate modification by dehydratases from type I polyketide synthases Journal Article In: 2017. @article{noKey,
title = {Insights into substrate modification by dehydratases from type I polyketide synthases},
author = {Faille, Alexandre, Gavalda, Sabine},
url = {https://doi.org/10.1016/j.jmb.2017.03.026},
doi = {http://dx.doi.org/10.1016/j.jmb.2017.03.026},
year = {2017},
date = {2017-03-27},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Mpakali, Anastasia, Saridakis, Emmanuel Ligand-induced conformational change of insulin-regulated aminopeptidase: Insights on catalytic mechanism and active site plasticity Journal Article In: 2017. @article{noKey,
title = {Ligand-induced conformational change of insulin-regulated aminopeptidase: Insights on catalytic mechanism and active site plasticity},
author = {Mpakali, Anastasia, Saridakis, Emmanuel},
url = {https://ora.ox.ac.uk/objects/uuid:e215f1e8-386b-42d8-a818-a0461d9ea763/download_file?safe_filename=Mpakali-Greece-JMC-2017.pdf&file_format=application%2Fpdf&type_of_work=Journal+article},
doi = {10.1021/acs.jmedchem.6b01890.},
year = {2017},
date = {2017-03-22},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Salvati Manni, Livia Design, Characterization and Biomedical Applications of Cyclopropanated Lipidic Mesophases Journal Article In: 2017. @article{noKey,
title = {Design, Characterization and Biomedical Applications of Cyclopropanated Lipidic Mesophases},
author = {Salvati Manni, Livia},
url = {https://www.zora.uzh.ch/id/eprint/147198/8/20183239.pdf},
doi = {https://doi.org/10.5167/uzh-147198},
year = {2017},
date = {2017-03-20},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Morgenstern, Josefine, Baumann, Pascal Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme Journal Article In: 2017. @article{noKey,
title = {Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme},
author = {Morgenstern, Josefine, Baumann, Pascal},
url = {https://www.sciencedirect.com/science/article/abs/pii/S0378517317300492?via%3Dihub},
doi = {https://doi.org/10.1016/j.ijpharm.2017.01.040},
year = {2017},
date = {2017-03-15},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Deibler, Kristine K., Mishra, Rama K. A Chemical Probe Strategy for Interrogating Inhibitor Selectivity Across the MEK Kinase Family Journal Article In: 2017. @article{noKey,
title = {A Chemical Probe Strategy for Interrogating Inhibitor Selectivity Across the MEK Kinase Family},
author = {Deibler, Kristine K., Mishra, Rama K.},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652073/},
doi = {https://doi.org/10.1021/acschembio.6b01060},
year = {2017},
date = {2017-03-06},
abstract = {MEK4 is an upstream kinase in MAPK signaling pathways where it phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Despite a high level of sequence homology in the ATP-binding site, most reported MEK inhibitors are selective for MEK1/2 and display reduced potency toward other MEKs. Here, we present the first functional and binding selectivity-profiling platform of the MEK family. We applied the platform to profile a set of known kinase inhibitors and used the results to develop an in silico approach for small molecule docking against MEK proteins. The docking studies identified molecular features of the ligands and corresponding amino acids in MEK proteins responsible for high affinity binding versus those driving selectivity. WaterLOGSY and saturation transfer difference (STD) NMR spectroscopy techniques were utilized to understand the binding modes of active compounds. Further minor synthetic manipulations provide a proof of concept by showing how information gained through this platform can be utilized to perturb selectivity across the MEK family. This inhibitor-based approach pinpoints key features governing MEK family selectivity and clarifies empirical selectivity profiles for a set of kinase inhibitors. Going forward, the platform provides a rationale for facilitating the development of MEK-selective inhibitors, particularly MEK4 selective inhibitors, and repurposing of kinase inhibitors for probing the structural selectivity of isoforms.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
MEK4 is an upstream kinase in MAPK signaling pathways where it phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Despite a high level of sequence homology in the ATP-binding site, most reported MEK inhibitors are selective for MEK1/2 and display reduced potency toward other MEKs. Here, we present the first functional and binding selectivity-profiling platform of the MEK family. We applied the platform to profile a set of known kinase inhibitors and used the results to develop an in silico approach for small molecule docking against MEK proteins. The docking studies identified molecular features of the ligands and corresponding amino acids in MEK proteins responsible for high affinity binding versus those driving selectivity. WaterLOGSY and saturation transfer difference (STD) NMR spectroscopy techniques were utilized to understand the binding modes of active compounds. Further minor synthetic manipulations provide a proof of concept by showing how information gained through this platform can be utilized to perturb selectivity across the MEK family. This inhibitor-based approach pinpoints key features governing MEK family selectivity and clarifies empirical selectivity profiles for a set of kinase inhibitors. Going forward, the platform provides a rationale for facilitating the development of MEK-selective inhibitors, particularly MEK4 selective inhibitors, and repurposing of kinase inhibitors for probing the structural selectivity of isoforms. |
Schaaf, Tory M., Peterson, Kurt C Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells Journal Article In: 2017. @article{noKey,
title = {Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells},
author = {Schaaf, Tory M., Peterson, Kurt C},
url = {https://journals.sagepub.com/doi/full/10.1177/1087057116679637},
doi = {https://doi.org/10.1177/1087057116679637},
year = {2017},
date = {2017-03-01},
abstract = {We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery. |
Wiktoria Ogrodowicz, Roksana Studies on low pH-activated HA2 from Influenza haemagglutinin Journal Article In: 2017. @article{noKey,
title = {Studies on low pH-activated HA2 from Influenza haemagglutinin},
author = {Wiktoria Ogrodowicz, Roksana},
url = {https://discovery.ucl.ac.uk/id/eprint/1540872/1/FINAL_RPS_REDUCED.pdf},
doi = {null},
year = {2017},
date = {2017-02-28},
abstract = {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.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
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. |
Melo, Arthur Alves, Hegde, Balachandra G. Structural insights into the activation mechanism of dynamin-like EHD ATPases Journal Article In: 2017. @article{noKey,
title = {Structural insights into the activation mechanism of dynamin-like EHD ATPases},
author = {Melo, Arthur Alves, Hegde, Balachandra G.},
url = {https://www.pnas.org/content/114/22/5629},
doi = {https://doi.org/10.1073/pnas.1614075114},
year = {2017},
date = {2017-02-22},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Melo, Arthur Structural insights into the activation mechanism of the EHD family Journal Article In: 2017. @article{noKey,
title = {Structural insights into the activation mechanism of the EHD family},
author = {Melo, Arthur},
url = {https://refubium.fu-berlin.de/bitstream/handle/fub188/6484/Melo_thesis_final.pdf?sequence=1&isAllowed=y},
doi = {null},
year = {2017},
date = {2017-02-22},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Ghachi, Meriem El, Howe, Nicole Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis Journal Article In: 2017. @article{noKey,
title = {Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis},
author = {Ghachi, Meriem El, Howe, Nicole},
url = {https://link.springer.com/article/10.1007%2Fs00018-017-2464-6},
doi = {https://doi.org/10.1007/s00018-017-2464-6},
year = {2017},
date = {2017-02-06},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Wright, Jack, Thomsen, Maren The crystal structure of PD1, a Haemophilus surface fibril domain Journal Article In: 2017. @article{noKey,
title = {The crystal structure of PD1, a Haemophilus surface fibril domain},
author = {Wright, Jack, Thomsen, Maren},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297931/},
doi = {https://doi.org/10.1107%2FS2053230X17001406},
year = {2017},
date = {2017-02-01},
abstract = {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.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
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. |
McElwain, Mark A., Yu Zhang, Rebecca Long Fragment Read (LFR) Technology: Cost-Effective, High-Quality Genome-Wide Molecular Haplotyping Journal Article In: 2017. @article{noKey,
title = {Long Fragment Read (LFR) Technology: Cost-Effective, High-Quality Genome-Wide Molecular Haplotyping},
author = {McElwain, Mark A., Yu Zhang, Rebecca},
url = {https://link.springer.com/protocol/10.1007/978-1-4939-6750-6_11},
doi = {https://doi.org/10.1007/978-1-4939-6750-6_11},
year = {2017},
date = {2017-01-31},
abstract = {In this chapter, we describe Long Fragment Read (LFR) technology, a DNA preprocessing method for genome-wide haplotyping by whole genome sequencing (WGS). The addition of LFR prior to WGS on any high-throughput DNA sequencer (e.g., Complete Genomics Revolocity™, BGISEQ-500, Illumina HiSeq, etc.) enables the assignment of single-nucleotide polymorphisms (SNPs) and other genomic variants onto contigs representing contiguous DNA from a single parent (haplotypes) with N50 lengths of up to ~1 Mb. Importantly, this is achieved independent of any parental sequencing data or knowledge of parental haplotypes. Further, the nature of this method allows for the correction of most amplification, sequencing, and mapping errors, resulting in false-positive error rates as low as 10−9. This method can be employed either manually using hand-held micropipettors or in the preferred, automated manner described below, utilizing liquid-handling robots capable of pipetting in the nanoliter range. Automating the method limits the amount of hands-on time and allows significant reduction in reaction volumes. Further, the cost of LFR, as described in this chapter, is moderate, while it adds invaluable whole genome haplotype data to almost any WGS process.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
In this chapter, we describe Long Fragment Read (LFR) technology, a DNA preprocessing method for genome-wide haplotyping by whole genome sequencing (WGS). The addition of LFR prior to WGS on any high-throughput DNA sequencer (e.g., Complete Genomics Revolocity™, BGISEQ-500, Illumina HiSeq, etc.) enables the assignment of single-nucleotide polymorphisms (SNPs) and other genomic variants onto contigs representing contiguous DNA from a single parent (haplotypes) with N50 lengths of up to ~1 Mb. Importantly, this is achieved independent of any parental sequencing data or knowledge of parental haplotypes. Further, the nature of this method allows for the correction of most amplification, sequencing, and mapping errors, resulting in false-positive error rates as low as 10−9. This method can be employed either manually using hand-held micropipettors or in the preferred, automated manner described below, utilizing liquid-handling robots capable of pipetting in the nanoliter range. Automating the method limits the amount of hands-on time and allows significant reduction in reaction volumes. Further, the cost of LFR, as described in this chapter, is moderate, while it adds invaluable whole genome haplotype data to almost any WGS process. |
Hämmerling, Frank, Lorenz-Cristea, Oliver Strategy for assessment of the colloidal and biological stability of H1N1 influenza A viruses Journal Article In: 2017. @article{noKey,
title = {Strategy for assessment of the colloidal and biological stability of H1N1 influenza A viruses},
author = {Hämmerling, Frank, Lorenz-Cristea, Oliver},
url = {https://www.sciencedirect.com/science/article/abs/pii/S0378517316311218?via%3Dihub},
doi = {https://doi.org/10.1016/j.ijpharm.2016.11.058},
year = {2017},
date = {2017-01-30},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Nicolussi, Andrea, Dunn, Joe Dan Secreted heme peroxidase from Dictyostelium discoideum: Insights into catalysis, structure, and biological role Journal Article In: 2017. @article{noKey,
title = {Secreted heme peroxidase from Dictyostelium discoideum: Insights into catalysis, structure, and biological role},
author = {Nicolussi, Andrea, Dunn, Joe Dan},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787809/},
doi = {https://doi.org/10.1074/jbc.RA117.000463},
year = {2017},
date = {2017-01-26},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Nikolaev, M., Round, E. Integral Membrane Proteins Can Be Crystallized Directly from Nanodiscs Journal Article In: 2017. @article{noKey,
title = {Integral Membrane Proteins Can Be Crystallized Directly from Nanodiscs},
author = {Nikolaev, M., Round, E.},
url = {https://pubs.acs.org/doi/full/10.1021/acs.cgd.6b01631},
doi = {https://doi.org/10.1021/acs.cgd.6b01631},
year = {2017},
date = {2017-01-20},
abstract = {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.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
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. |
Stagno, J. R., Liu, Y. Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography Journal Article In: 2017. @article{noKey,
title = {Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography},
author = {Stagno, J. R., Liu, Y.},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502819/},
doi = {https://doi.org/10.1038/nature20599},
year = {2017},
date = {2017-01-12},
abstract = {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.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
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. |
Sluchanko, Nikolai N., Beelen, Steven Structural basis for the interaction of a human small heat shock protein with the 14-3-3 universal signaling regulator Journal Article In: 2017. @article{noKey,
title = {Structural basis for the interaction of a human small heat shock protein with the 14-3-3 universal signaling regulator},
author = {Sluchanko, Nikolai N., Beelen, Steven},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321513/},
doi = {https://doi.org/10.1016/j.str.2016.12.005},
year = {2017},
date = {2017-01-12},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Chernyatina, Anastasia A., Hess, John F. How to Study Intermediate Filaments in Atomic Detail Journal Article In: 2016. @article{noKey,
title = {How to Study Intermediate Filaments in Atomic Detail},
author = {Chernyatina, Anastasia A., Hess, John F.},
url = {https://www.sciencedirect.com/science/article/abs/pii/S007668791500542X?via%3Dihub},
doi = {https://doi.org/10.1016/bs.mie.2015.09.024},
year = {2016},
date = {2016-12-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Santos, Raqueldos, Carvalho, Ana Luísa Renaissance of protein crystallization and precipitation in biopharmaceuticals purification Journal Article In: 2016. @article{noKey,
title = {Renaissance of protein crystallization and precipitation in biopharmaceuticals purification},
author = {Santos, Raqueldos, Carvalho, Ana Luísa},
url = {https://www.sciencedirect.com/science/article/abs/pii/S0734975016301513?via%3Dihub},
doi = {https://doi.org/10.1016/j.biotechadv.2016.11.005},
year = {2016},
date = {2016-11-28},
abstract = {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.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
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. |
Bauer, Katharina Christin, Suhm, Susanna Impact of additives on the formation of protein aggregates and viscosity in concentrated protein solutions Journal Article In: 2016. @article{noKey,
title = {Impact of additives on the formation of protein aggregates and viscosity in concentrated protein solutions},
author = {Bauer, Katharina Christin, Suhm, Susanna},
url = {https://pubmed.ncbi.nlm.nih.gov/27836754/},
doi = {https://doi.org/10.1016/j.ijpharm.2016.11.009},
year = {2016},
date = {2016-11-08},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
