scholarly journals Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zachary Maben ◽  
Richa Arya ◽  
Dimitris Georgiadis ◽  
Efstratios Stratikos ◽  
Lawrence J. Stern
Keyword(s):  
Conformational Dynamics ◽  
Allosteric Regulation ◽  
The Body ◽  
Regulation Mechanism ◽  
Antigenic Peptides ◽  
Mhc I ◽  
Peptide Substrates ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering

AbstractThe endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.

scholarly journals Structure of the Lassa Virus Nucleoprotein Revealed by X-ray Crystallography, Small-angle X-ray Scattering, and Electron Microscopy

2011 ◽  
Vol 286 (44) ◽  
pp. 38748-38756 ◽  
Author(s):  
Linda Brunotte ◽  
Romy Kerber ◽  
Weifeng Shang ◽  
Florian Hauer ◽  
Meike Hass ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Small Angle ◽  
Lassa Virus ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering ◽  
Ray Scattering

scholarly journals Exploiting the full quantum crystallography

2018 ◽  
Vol 96 (7) ◽  
pp. 599-605 ◽  
Author(s):  
Lou Massa ◽  
Chérif F. Matta
Keyword(s):  
Quantum Mechanics ◽  
Electron Density ◽  
Mathematical Structure ◽  
Scattering Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering ◽  
Fundamental Value ◽  
Quantum Crystallography ◽  
Ray Scattering

Quantum crystallography (QCr) is a branch of crystallography aimed at obtaining the complete quantum mechanics of a crystal given its X-ray scattering data. The fundamental value of obtaining an electron density matrix that is N-representable is that it ensures consistency with an underlying properly antisymmetrized wavefunction, a requirement of quantum mechanical validity. However, X-ray crystallography has progressed in an impressive way for decades based only upon the electron density obtained from the X-ray scattering data without the imposition of the mathematical structure of quantum mechanics. Therefore, one may perhaps ask regarding N-representability “why bother?” It is the purpose of this article to answer such a question by succinctly describing the advantage that is opened by QCr.

scholarly journals A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I

2020 ◽  
Vol 117 (50) ◽  
pp. 31838-31849
Author(s):  
Rebecca Ebenhoch ◽  
Simone Prinz ◽  
Susann Kaltwasser ◽  
Deryck J. Mills ◽  
Robert Meinecke ◽  
...  
Keyword(s):  
Crystal Packing ◽  
Substrate Binding ◽  
Regulatory Protein ◽  
Allosteric Regulation ◽  
Hybrid Approach ◽  
Site Directed Mutagenesis ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
X Ray ◽  
X Ray Crystallography

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

scholarly journals Integrative Approaches in Structural Biology: A More Complete Picture from the Combination of Individual Techniques

Biomolecules ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 370 ◽  
Author(s):  
Linda Cerofolini ◽  
Marco Fragai ◽  
Enrico Ravera ◽  
Christoph A. Diebolder ◽  
Ludovic Renault ◽  
...  
Keyword(s):  
Structural Biology ◽  
Catalytic Mechanism ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering ◽  
Protein Model ◽  
Pros And Cons ◽  
Transient Interactions ◽  
Single Technique ◽  
Nmr Restraints

With the recent technological and computational advancements, structural biology has begun to tackle more and more difficult questions, including complex biochemical pathways and transient interactions among macromolecules. This has demonstrated that, to approach the complexity of biology, one single technique is largely insufficient and unable to yield thorough answers, whereas integrated approaches have been more and more adopted with successful results. Traditional structural techniques (X-ray crystallography and Nuclear Magnetic Resonance (NMR)) and the emerging ones (cryo-electron microscopy (cryo-EM), Small Angle X-ray Scattering (SAXS)), together with molecular modeling, have pros and cons which very nicely complement one another. In this review, three examples of synergistic approaches chosen from our previous research will be revisited. The first shows how the joint use of both solution and solid-state NMR (SSNMR), X-ray crystallography, and cryo-EM is crucial to elucidate the structure of polyethylene glycol (PEG)ylated asparaginase, which would not be obtainable through any of the techniques taken alone. The second deals with the integrated use of NMR, X-ray crystallography, and SAXS in order to elucidate the catalytic mechanism of an enzyme that is based on the flexibility of the enzyme itself. The third one shows how it is possible to put together experimental data from X-ray crystallography and NMR restraints in order to refine a protein model in order to obtain a structure which simultaneously satisfies both experimental datasets and is therefore closer to the ‘real structure’.

scholarly journals Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering

2014 ◽  
Vol 111 (50) ◽  
pp. 17887-17892 ◽  
Author(s):  
Michael E. Wall ◽  
Andrew H. Van Benschoten ◽  
Nicholas K. Sauter ◽  
Paul D. Adams ◽  
James S. Fraser ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Conformational Dynamics ◽  
X Ray ◽  
X Ray Scattering ◽  
Dynamics Simulations ◽  
Ray Scattering

Regulative interactions of the osmosensing C-terminal domain in the trimeric glycine betaine transporter BetP from Corynebacterium glutamicum

2009 ◽  
Vol 390 (8) ◽  
Author(s):  
Reinhard Krämer ◽  
Christine Ziegler
Keyword(s):  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Protein Interactions ◽  
Regulation Mechanism ◽  
Protein Protein Interactions ◽  
Molecular Switching ◽  
X Ray ◽  
X Ray Crystallography ◽  
Terminal Domain ◽  
Domain Reorientation

Abstract Activation of the osmoregulated trimeric betaine transporter BetP from Corynebacterium glutamicum was shown to depend mainly on the correct folding and integrity of its 55 amino acid long, partly α-helical C-terminal domain. Reorientation of the three C-terminal domains in the BetP trimer indicates different lipid-protein and protein-protein interactions of the C-terminal domain during osmoregulation. A regulation mechanism is suggested where this domain switches the transporter from the inactive to the active state. Interpretation of recently obtained electron and X-ray crystallography data of BetP led to a structure-function based model of C-terminal molecular switching involved in osmoregulation.

High-pressure developments for resonant X-ray scattering experiments at I16

2020 ◽  
Vol 27 (2) ◽  
pp. 351-359
Author(s):  
I. Povedano ◽  
A. Bombardi ◽  
D. G. Porter ◽  
M. Burt ◽  
S. Green ◽  
...  
Keyword(s):  
High Pressure ◽  
Low Temperature ◽  
The Body ◽  
X Ray ◽  
X Ray Scattering ◽  
Scattering Geometry ◽  
Diamond Anvil ◽  
Membrane Cell ◽  
Ray Scattering

An experimental setup to perform high-pressure resonant X-ray scattering (RXS) experiments at low temperature on I16 at Diamond Light Source is presented. The setup consists of a membrane-driven diamond anvil cell, a panoramic dome and an optical system that allows pressure to be measured in situ using the ruby fluorescence method. The membrane cell, inspired by the Merrill–Bassett design, presents an asymmetric layout in order to operate in a back-scattering geometry, with a panoramic aperture of 100° in the top and a bottom half dedicated to the regulation and measurement of pressure. It is specially designed to be mounted on the cold finger of a 4 K closed-cycle cryostat and actuated at low-temperature by pumping helium into the gas membrane. The main parts of the body are machined from a CuBe alloy (BERYLCO 25) and, when assembled, it presents an approximate height of 20–21 mm and fits into a 57 mm diameter. This system allows different materials to be probed using RXS in a range of temperatures between 30 and 300 K and has been tested up to 20 GPa using anvils with a culet diameter of 500 µm under quasi-cryogenic conditions. Detailed descriptions of different parts of the setup, operation and the developed methodology are provided here, along with some preliminary experimental results.

scholarly journals From lows to highs: using low-resolution models to phase X-ray data

2013 ◽  
Vol 69 (11) ◽  
pp. 2257-2265 ◽  
Author(s):  
David I. Stuart ◽  
Nicola G. A. Abrescia
Keyword(s):  
Structural Biology ◽  
General Concept ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering ◽  
Phase Extension ◽  
Intact Virus ◽  
Structural Methods

The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ∼13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.

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