scholarly journals Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution

2021 ◽  
Vol 12 ◽  
Author(s):  
Monica L. Fernández-Quintero ◽  
Katharina B. Kroell ◽  
Florian Hofer ◽  
Jakob R. Riccabona ◽  
Klaus R. Liedl
Keyword(s):  
Conformational Changes ◽  
Antibody Engineering ◽  
Antigen Binding Site ◽  
Binding Interface ◽  
Loop Conformations ◽  
Functional Consequences ◽  
Complementarity Determining Regions ◽  
Antibody Design

Characterizing and understanding the antibody binding interface have become a pre-requisite for rational antibody design and engineering. The antigen-binding site is formed by six hypervariable loops, known as the complementarity determining regions (CDRs) and by the relative interdomain orientation (VH–VL). Antibody CDR loops with a certain sequence have been thought to be limited to a single static canonical conformation determining their binding properties. However, it has been shown that antibodies exist as ensembles of multiple paratope states, which are defined by a characteristic combination of CDR loop conformations and interdomain orientations. In this study, we thermodynamically and kinetically characterize the prominent role of residue 71H (Chothia nomenclature), which does not only codetermine the canonical conformation of the CDR-H2 loop but also results in changes in conformational diversity and population shifts of the CDR-H1 and CDR-H3 loop. As all CDR loop movements are correlated, conformational rearrangements of the heavy chain CDR loops also induce conformational changes in the CDR-L1, CDR-L2, and CDR-L3 loop. These overall conformational changes of the CDR loops also influence the interface angle distributions, consequentially leading to different paratope states in solution. Thus, the type of residue of 71H, either an alanine or an arginine, not only influences the CDR-H2 loop ensembles, but co-determines the paratope states in solution. Characterization of the functional consequences of mutations of residue 71H on the paratope states and interface orientations has broad implications in the field of antibody engineering.

scholarly journals Evidence that the TRPV1 S1-S4 Membrane Domain Contributes to Thermosensing

2019 ◽  
Author(s):  
Minjoo Kim ◽  
Nicholas J. Sisco ◽  
Jacob K. Hilton ◽  
Camila M. Montano ◽  
Manuel A. Castro ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Solution Nmr ◽  
Membrane Domain ◽  
Temperature Dependent ◽  
Nmr Characterization ◽  
Significant Interest ◽  
Pore Domain ◽  
Coupling Mechanisms

AbstractSensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. However, the role of the S1-S4 domain in thermosensing is unclear. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, Far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

scholarly journals Structural and Dynamic Characterizations Highlight the Deleterious Role of SULT1A1 R213H Polymorphism in Substrate Binding

2019 ◽  
Vol 20 (24) ◽  
pp. 6256 ◽  
Author(s):  
Raju Dash ◽  
Md. Chayan Ali ◽  
Nayan Dash ◽  
Md. Abul Kalam Azad ◽  
S. M. Zahid Hosen ◽  
...  
Keyword(s):  
Binding Energy ◽  
Cancer Progression ◽  
Conformational Changes ◽  
Substrate Binding ◽  
Dynamics Simulation ◽  
Loss Of Function ◽  
Functional Consequences ◽  
Binding Energy Analysis ◽  
Exogenous Compounds

Sulfotransferase 1A1 (SULT1A1) is responsible for catalyzing various types of endogenous and exogenous compounds. Accumulating data indicates that the polymorphism rs9282861 (R213H) is responsible for inefficient enzymatic activity and associated with cancer progression. To characterize the detailed functional consequences of this mutation behind the loss-of-function of SULT1A1, the present study deployed molecular dynamics simulation to get insights into changes in the conformation and binding energy. The dynamics scenario of SULT1A1 in both wild and mutated types as well as with and without ligand showed that R213H induced local conformational changes, especially in the substrate-binding loop rather than impairing overall stability of the protein structure. The higher conformational changes were observed in the loop3 (residues, 235–263), turning loop conformation to A-helix and B-bridge, which ultimately disrupted the plasticity of the active site. This alteration reduced the binding site volume and hydrophobicity to decrease the binding affinity of the enzyme to substrates, which was highlighted by the MM-PBSA binding energy analysis. These findings highlight the key insights of structural consequences caused by R213H mutation, which would enrich the understanding regarding the role of SULT1A1 mutation in cancer development and also xenobiotics management to individuals in the different treatment stages.

scholarly journals Allosteric switch regulates protein–protein binding through collective motion

2016 ◽  
Vol 113 (12) ◽  
pp. 3269-3274 ◽  
Author(s):  
Colin A. Smith ◽  
David Ban ◽  
Supriya Pratihar ◽  
Karin Giller ◽  
Maria Paulat ◽  
...  
Keyword(s):  
Collective Motion ◽  
Protein Domain ◽  
Allosteric Coupling ◽  
Allosteric Communication ◽  
Binding Interface ◽  
Specific Protease ◽  
Ubiquitin Specific Protease ◽  
Promising Avenue

Many biological processes depend on allosteric communication between different parts of a protein, but the role of internal protein motion in propagating signals through the structure remains largely unknown. Through an experimental and computational analysis of the ground state dynamics in ubiquitin, we identify a collective global motion that is specifically linked to a conformational switch distant from the binding interface. This allosteric coupling is also present in crystal structures and is found to facilitate multispecificity, particularly binding to the ubiquitin-specific protease (USP) family of deubiquitinases. The collective motion that enables this allosteric communication does not affect binding through localized changes but, instead, depends on expansion and contraction of the entire protein domain. The characterization of these collective motions represents a promising avenue for finding and manipulating allosteric networks.

scholarly journals Structure and inhibitor binding characterization of oncogenic MLLT1 mutants

2021 ◽  
Author(s):  
Xiaomin Ni ◽  
Allyn T. Londregan ◽  
Dafydd R. Owen ◽  
Stefan Knapp ◽  
Apirat Chaikuad
Keyword(s):  
Conformational Changes ◽  
Structural Basis ◽  
Wild Type ◽  
Structural Comparison ◽  
Inhibitor Binding ◽  
Binding Interface ◽  
Association Behavior ◽  
Self Association ◽  
Potential Strategy

AbstractDysfunction of YEATS-domain-containing MLLT1, an acetyl/acyl-lysine dependent epigenetic reader domain, has been implicated in the development of aggressive cancers. Mutations in the YEATS domain have been recently reported as a cause of MLLT1 aberrant reader function. However, structural basis for the reported alterations in affinity for acetyled/acylated histone has remained elusive. Here, we report the crystal structures of both insertion and substitution present in cancer, revealing significant conformational changes of the YEATS-domain loop 8. Structural comparison demonstrates that such alteration not only altered the binding interface for acetylated/acylated histones, but the sequence alterations in the T1 loop may enable dimeric assembly consistent inducing self-association behavior. Nevertheless, we show that also the MLLT1 mutants can be targeted by developed acetyllysine mimetic inhibitors with affinities similarly to wild type. Our report provides a structural basis for the altered behaviors and potential strategy for targeting oncogenic MLLT1 mutants.

scholarly journals Conformational selection of allergen-antibody complexes—surface plasticity of paratopes and epitopes

2019 ◽  
Vol 32 (11) ◽  
pp. 513-523
Author(s):  
Monica L Fernández-Quintero ◽  
Johannes R Loeffler ◽  
Franz Waibl ◽  
Anna S Kamenik ◽  
Florian Hofer ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Information ◽  
High Surface ◽  
Epitope Prediction ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
Surface Plasticity ◽  
Structural Framework ◽  
Dynamics Simulations

Abstract Antibodies have the ability to bind various types of antigens and to recognize different antibody-binding sites (epitopes) of the same antigen with different binding affinities. Due to the conserved structural framework of antibodies, their specificity to antigens is mainly determined by their antigen-binding site (paratope). Therefore, characterization of epitopes in combination with describing the involved conformational changes of the paratope upon binding is crucial in understanding and predicting antibody-antigen binding. Using molecular dynamics simulations complemented with strong experimental structural information, we investigated the underlying binding mechanism and the resulting local and global surface plasticity in the binding interfaces of distinct antibody-antigen complexes. In all studied allergen-antibody complexes, we clearly observe that experimentally suggested epitopes reveal less plasticity, while non-epitope regions show high surface plasticity. Surprisingly, the paratope shows higher conformational diversity reflected in substantially higher surface plasticity, compared to the epitope. This work allows a visualization and characterization of antibody-antigen interfaces and might have strong implications for antibody-antigen docking and in the area of epitope prediction.

scholarly journals Role of the S3-S4 Linker in Shaker Potassium Channel Activation

1997 ◽  
Vol 109 (2) ◽  
pp. 191-199 ◽  
Author(s):  
Rajesh Mathur ◽  
Jie Zheng ◽  
Yangyang Yan ◽  
Fred J. Sigworth
Keyword(s):  
Conformational Changes ◽  
Xenopus Oocytes ◽  
Channel Activation ◽  
Activation Kinetics ◽  
Shaker Potassium Channel ◽  
Large Conformational Change ◽  
Voltage Gated Channels ◽  
Mutant Construct

Structural models of voltage-gated channels suggest that flexibility of the S3-S4 linker region may be important in allowing the S4 region to undergo large conformational changes in its putative voltage-sensing function. We report here the initial characterization of 18 mutations in the S3-S4 linker of the Shaker channel, including deletions, insertions, charge changes, substitution of prolines, and chimeras replacing the 25-residue Shaker linker with 7- or 9-residue sequences from Shab, Shaw, or Shal. As measured in Xenopus oocytes with a two-microelectrode voltage clamp, each mutant construct yielded robust currents. Changes in the voltage dependence of activation were small, with activation voltage shifts of 13 mV or less. Substitution of linkers from the slowly activating Shab and Shaw channels resulted in a three- to fourfold slowing of activation and deactivation. It is concluded that the S3-S4 linker is unlikely to participate in a large conformational change during channel activation. The linker, which in some channel subfamilies has highly conserved sequences, may however be a determinant of activation kinetics in potassium channels, as previously has been suggested in the case of calcium channels.

scholarly journals Characterization of Large Conformational Changes and Autoproteolysis in the Maturation of a T=4 Virus Capsid

2008 ◽  
Vol 83 (2) ◽  
pp. 1126-1134 ◽  
Author(s):  
Tsutomu Matsui ◽  
Gabriel Lander ◽  
John E. Johnson
Keyword(s):  
Conformational Changes ◽  
Cleavage Site ◽  
Cleavage Rate ◽  
Virus Particles ◽  
Wild Type Phenotype ◽  
Optimum Ph ◽  
Reaction Proceeds ◽  
Intermediate Size

ABSTRACT Nudaurelia capensis ω virus-like particles have been characterized as a 480-Å procapsid and a 410-Å capsid, both with T=4 quasisymmetry. Procapsids transition to capsids when pH is lowered from 7.6 to 5.0. Capsids undergo autoproteolysis at residue 570, generating the 74-residue C-terminal polypeptide that remains with the particle. Here we show that the particle size becomes smaller under conditions between pH 6.8 and 6.0 without activating cleavage and that the particle remains at an intermediate size when the pH is carefully maintained. At pH 5.8, cleavage is very slow, becoming detectable only after 9 h. The optimum pH for cleavage is 5.0 (half-life, ∼30 min), with a significant reduction in the cleavage rate at pH values below 5. We also show that lowering the pH is required only to make the virus particles compact and to presumably form the active site for autoproteolysis but not for the chemistry of cleavage. The cleavage reaction proceeds at pH 7.0 after ∼10% of the subunits cleave at pH 5.0. Employing the virion crystal structure for reference, we investigated the role of electrostatic repulsion of acidic residues in the pH-dependent large conformational changes. Three mutations of Glu to Gln that formed procapsids showed three different phenotypes on maturation. One, close to the threefold and quasithreefold symmetry axes and far from the cleavage site, did not mature at pH 5, and electron cryomicroscopy reconstruction showed that it was intermediate in size between those of the procapsid and capsid; one near the cleavage site exhibited a wild-type phenotype; and a third, far from the cleavage site, resulted in cleavage of 50% of the subunits after 4 h, suggesting quasiequivalent specificity of the mutation.

scholarly journals Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations

2017 ◽  
Author(s):  
Rafael M. Couñago ◽  
Charles K. Allerston ◽  
Pavel Savitsky ◽  
Hatylas Azevedo ◽  
Paulo H. Godoi ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Related Protein ◽  
Chemical Probes ◽  
Cellular Processes ◽  
Starting Point ◽  
Loop Conformations ◽  
Potential Use ◽  
Specific Inhibitors

ABSTRACTThe human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.

scholarly journals The role of water molecules in the binding of class I and II peptides to the SH3 domain of the Fyn tyrosine kinase

2016 ◽  
Vol 72 (9) ◽  
pp. 707-712 ◽  
Author(s):  
Ana Camara-Artigas ◽  
Emilia Ortiz-Salmeron ◽  
Montserrrat Andujar-Sánchez ◽  
Julio Bacarizo ◽  
Jose Manuel Martin-Garcia
Keyword(s):  
Tyrosine Kinase ◽  
Conformational Changes ◽  
Sh3 Domain ◽  
Class I ◽  
Water Molecules ◽  
Src Tyrosine Kinase ◽  
Sh3 Domains ◽  
Binding Interface ◽  
Fyn Tyrosine Kinase

Interactions of proline-rich motifs with SH3 domains are present in signal transduction and other important cell processes. Analysis of structural and thermodynamic data suggest a relevant role of water molecules in these protein–protein interactions. To determine whether or not the SH3 domain of the Fyn tyrosine kinase shows the same behaviour, the crystal structures of its complexes with two high-affinity synthetic peptides, VSL12 and APP12, which are class I and II peptides, respectively, have been solved. In the class I complexes two water molecules were found at the binding interface that were not present in the class II complexes. The structures suggest a role of these water molecules in facilitating conformational changes in the SH3 domain to allow the binding of the class I or II peptides. In the third binding pocket these changes modify the cation–π and salt-bridge interactions that determine the affinity of the binding. Comparison of the water molecules involved in the binding of the peptides with previous reported hydration spots suggests a different pattern for the SH3 domains of the Src tyrosine kinase family.

Sign in / Sign up

Export Citation Format

Share Document