scholarly journals Recognition of histone H3 methylation states by the PHD1 domain of histone demethylase KDM5A

2019 ◽  
Author(s):  
James E Longbotham ◽  
Mark J S Kelly ◽  
Danica Galonić Fujimori
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Solution Structure ◽  
Structural Information ◽  
Protein Complexes ◽  
Histone H3 ◽  
Histone Demethylase ◽  
Helical Conformation ◽  
Regulatory Function ◽  
Small Molecule Modulators

AbstractPHD reader domains are chromatin binding modules often responsible for the recruitment of large protein complexes that contain histone modifying enzymes, chromatin remodelers and DNA repair machinery. A majority of PHD domains recognize N–terminal residues of histone H3 and are sensitive to the methylation state of Lys4 in histone H3 (H3K4). Histone demethylase KDM5A, an epigenetic eraser enzyme that contains three PHD domains, is often overexpressed in various cancers and its demethylation activity is allosterically enhanced when its PHD1 domain is bound to the H3 tail. The allosteric regulatory function of PHD1 expands roles of reader domains, suggesting unique features of this chromatin interacting module. Our previous studies determined the H3 binding site of PHD1, although it remains unclear how the H3 tail interacts with the N–terminal residues of PHD1 and how PHD1 discriminates against H3 tails with varying degrees of H3K4 methylation. Here we have determined the solution structure of apo and H3 bound PHD1. We observe conformational changes occurring in PHD1 in order to accommodate H3, which interestingly binds in a helical conformation. We also observe differential interactions of binding residues with differently methylated H3K4 peptides (me0, me1, me2 or me3), providing a rational for this PHD1 domain’s preference for lower methylation states of H3K4. We further assessed the contributions of various H3 interacting residues in the PHD1 domain to the binding of H3 peptides. The structural information of the H3 binding site could provide useful information to aid development of allosteric small molecule modulators of KDM5A.

scholarly journals Structural and energetic aspects of protein-protein recognition.

1997 ◽  
Vol 44 (3) ◽  
pp. 367-387 ◽  
Author(s):  
J Otlewski ◽  
W Apostoluk
Keyword(s):  
Conformational Changes ◽  
Growth Hormone Receptor ◽  
Structural Information ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Water Structure ◽  
Human Growth ◽  
Protein Recognition ◽  
Specific Recognition ◽  
Different Types

Specific recognition between proteins plays a crucial role in a great number of vital processes. In this review different types of protein-protein complexes are analyzed on the basis of their three-dimensional structures which became available in recent years. The complexes which are analyzed include: those resulting from different types of recognition between proteinase and protein inhibitor (canonical inhibitors of serine proteinases, hirudin, inhibitors of cysteine proteinases, carboxypeptidase inhibitor), barnase-barstar, human growth hormone-receptor and antibody-antigen. It seems obvious that specific and strong protein-protein recognition is achieved in many different ways. To further explore this question, the structural information was analyzed together with kinetic and thermodynamic data available for the respective complexes. It appears that the energy and rates of specific recognition of proteins are influenced by many different factors, including: area of interacting surfaces; complementarity of shapes, charges and hydrogen bonds; water structure at the interface; conformational changes; additivity and cooperativity of individual interactions, steric effects and various (conformational, hydration) entropy changes.

scholarly journals An enhanced-sampling MD-based protocol for molecular docking

2018 ◽  
Author(s):  
Andrea Basciu ◽  
Giuliano Malloci ◽  
Fabio Pietrucci ◽  
Alexandre M. J. J. Bonvin ◽  
Attilio V. Vargiu
Keyword(s):  
Molecular Dynamics ◽  
Molecular Docking ◽  
Drug Design ◽  
Binding Site ◽  
Conformational Changes ◽  
Protein Complexes ◽  
Structural Diversity ◽  
Collective Variables ◽  
Structure Based Drug Design ◽  
Ensemble Docking

AbstractUnderstanding molecular recognition of proteins by small molecules is key for drug design. Despite the number of experimental structures of ligand-protein complexes keeps growing, the number of available targets remains limited compared to the druggable genome, and structural diversity is generally low, which affects the chemical variance of putative lead compounds. From a computational perspective, molecular docking is widely used to mimic ligand-protein association in silico. Ensemble-docking approaches include flexibility through a set of different conformations of the protein obtained either experimentally or from computer simulations, e.g. molecular dynamics. However, structures prone to host (the correct) ligands are generally poorly sampled by standard molecular dynamics simulations of the apo protein. In order to address this limitation, we introduce a computational approach based on metadynamics simulations (EDES - Ensemble-Docking with Enhanced-sampling of pocket Shape) to generate druggable conformations of proteins only exploiting their apo structures. This is achieved by defining a set of collective variables that effectively sample different shapes of the binding site, ultimately mimicking the steric effect due to ligands to generate holo-like binding site geometries. We assessed the method on two challenging proteins undergoing different extents of conformational changes upon ligand binding. In both cases our protocol generated a significant fraction of structures featuring a low RMSD from the experimental holo conformation. Moreover, ensemble docking calculations using those conformations yielded native-like poses among the top ranked ones for both targets. This proof of concept study paves the route towards an automated workflow to generate druggable conformations of proteins, which should become a precious tool for structure-based drug design.

Global conformational changes in IgG-Fc upon mutation of the FcRn-binding site are not associated with altered antibody-dependent effector functions

2018 ◽  
Vol 475 (13) ◽  
pp. 2179-2190
Author(s):  
Ingrid J.G. Burvenich ◽  
William Farrugia ◽  
Zhanqi Liu ◽  
Dahna Makris ◽  
Dylan King ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Solution Structure ◽  
Antibody Engineering ◽  
Target Cells ◽  
Wild Type ◽  
Gamma Receptor ◽  
Complement Dependent Cytotoxicity ◽  
Conformational Plasticity

Antibody engineering is important for many diagnostic and clinical applications of monoclonal antibodies. We recently reported a series of fragment crystallizable (Fc) mutations targeting the neonatal Fc receptor (FcRn) site on a Lewis Y (Ley) binding IgG1, hu3S193. The hu3S193 variants displayed shortened in vivo half-lives and may have potential for radioimaging or radiotherapy of Ley-positive tumors. Here, we report Fc crystal structures of wild-type hu3S193, seven FcRn-binding site variants, and a variant lacking C1q binding or complement-dependent cytotoxicity (CDC) activity. The Fc conformation of the FcRn-binding sites was similar for wild-type and all mutants of hu3S193 Fc, which suggests that FcRn interactions were directly affected by the amino acid substitutions. The C1q-binding site mutant Fc was nearly identical with the wild-type Fc. Surprisingly, several hu3S193 Fc variants showed large changes in global structure compared with wild-type Fc. All hu3S193 Fc mutants had similar antibody-dependent cellular cytotoxicity, despite some with conformations expected to diminish Fc gamma receptor binding. Several hu3S193 variants displayed altered CDC, but there was no correlation with the different Fc conformations. All versions of hu3S193, except the C1q-binding site mutant, bound C1q, suggesting that the altered CDC of some variants could result from different propensities to form IgG hexamers after engaging Ley on target cells. Overall, our findings support the concept that the antibody Fc is both flexible and mobile in solution. Structure-based design approaches should take into account the conformational plasticity of the Fc when engineering antibodies with optimal effector properties.

scholarly journals Investigation of protein–protein interactions by isotope‒edited Fourier transformed infrared spectroscopy

2004 ◽  
Vol 18 (3) ◽  
pp. 397-406 ◽  
Author(s):  
Tiansheng Li
Keyword(s):  
Ftir Spectroscopy ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Isotope Labeling ◽  
Structural Information ◽  
Protein Complexes ◽  
Secondary Structures ◽  
Receptor Complex ◽  
Protein Protein Interactions

Recent advance in FTIR spectroscopy has shown the usefulness of13C uniform isotope labeling in proteins to study protein–protein interactions.13C uniform isotope labeling can significantly resolve the spectral overlap in the amide I/I′ region in the spectra of protein–protein complexes, and therefore allows more accurate determination of secondary structures of individual protein component in the complex than does the conventional FTIR spectroscopy. Only a limited number of biophysical techniques can be used effectively to obtain structural information of large protein–protein complex in solution. Though X‒ray crystallography and NMR have been used to provide structural information of proteins at atomic resolution, they are limited either by the ability of protein to crystallize or the large molecular weight of protein. Vibrational spectroscopy, including FTIR and Raman spectroscopies, has been extensively employed to investigate secondary structures and conformational dynamics of protein–protein complexes. However, significant spectral overlap in the amide I/Iʹ region in the spectra of protein–protein complexes often hinders the utilization of vibrational spectroscopy in the study of protein–protein complex. In this review, we shall discuss our recent work involving the application of isotope labeled FTIR to the investigation of protein–protein complexes such as cytokine–receptor complexes. One of the examples involves G‒CSF/receptor complex. To determine unambiguously the conformations of G‒CSF and the receptor in the complex, we have prepared uniformly13C/15N isotope labeled G‒CSF to resolve its amide Iʹ band from that of its receptor in the IR spectrum of the complex. Conformational changes and structural stability of individual protein subunit in G‒CSF/receptor complex have then been investigated by using FTIR spectroscopy (Li et al.,Biochemistry29 (1997), 8849–8859). Another example involves BDNF/trkB complex in which13C/15N uniformly labeled BDNF is complexed with its receptor trkB (Li et al.,Biopolymers67(1) (2002), 10–19). Interactions of13C/15N uniformly labeled brain‒derived neurotrophic factor (BDNF) with the extracellular domain of its receptor, trkB, have been investigated by employing FTIR spectroscopy. Conformational changes and structural stability and dynamics of BDNF/trkB complex have been determined unambiguously by FTIR spectroscopy, since amide I/Iʹ bands of13C/15N labeled BDNF are resolved from those of the receptor. Together, those studies have shown that isotope edited FTIR spectroscopy can be successfully applied to the determination of protein secondary structures of protein complexes containing either the same or different types of secondary structures. It was observed that13C/15N uniform labeling also affects significantly the frequency of amide IIʹ band, which may permit the determination of hydrogen–deuterium exchange in individual subunit of protein–protein complexes.

ATP-binding Cassette Exporters: Structure and Mechanism with a Focus on P-glycoprotein and MRP1

2019 ◽  
Vol 26 (7) ◽  
pp. 1062-1078 ◽  
Author(s):  
Maite Rocío Arana ◽  
Guillermo Alejandro Altenberg
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Structural Information ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
And Function ◽  
Atp Binding Cassette

Background:Proteins that belong to the ATP-binding cassette superfamily include transporters that mediate the efflux of substrates from cells. Among these exporters, P-glycoprotein and MRP1 are involved in cancer multidrug resistance, protection from endo and xenobiotics, determination of drug pharmacokinetics, and the pathophysiology of a variety of disorders. Objective:To review the information available on ATP-binding cassette exporters, with a focus on Pglycoprotein, MRP1 and related proteins. We describe tissue localization and function of these transporters in health and disease, and discuss the mechanisms of substrate transport. We also correlate recent structural information with the function of the exporters, and discuss details of their molecular mechanism with a focus on the nucleotide-binding domains. Methods: Evaluation of selected publications on the structure and function of ATP-binding cassette proteins. Conclusions:Conformational changes on the nucleotide-binding domains side of the exporters switch the accessibility of the substrate-binding pocket between the inside and outside, which is coupled to substrate efflux. However, there is no agreement on the magnitude and nature of the changes at the nucleotide- binding domains side that drive the alternate-accessibility. Comparison of the structures of Pglycoprotein and MRP1 helps explain differences in substrate selectivity and the bases for polyspecificity. P-glycoprotein substrates are hydrophobic and/or weak bases, and polyspecificity is explained by a flexible hydrophobic multi-binding site that has a few acidic patches. MRP1 substrates are mostly organic acids, and its polyspecificity is due to a single bipartite binding site that is flexible and displays positive charge.

scholarly journals Interaction between residues in the Mg2+-binding site regulates BK channel activation

2013 ◽  
Vol 141 (2) ◽  
pp. 217-228 ◽  
Author(s):  
Junqiu Yang ◽  
Huanghe Yang ◽  
Xiaohui Sun ◽  
Kelli Delaloye ◽  
Xiao Yang ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Structural Information ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activation ◽  
Cytosolic Domain ◽  
Membrane Spanning ◽  
Interdomain Interactions

As a unique member of the voltage-gated potassium channel family, a large conductance, voltage- and Ca2+-activated K+ (BK) channel has a large cytosolic domain that serves as the Ca2+ sensor, in addition to a membrane-spanning domain that contains the voltage-sensing (VSD) and pore-gate domains. The conformational changes of the cytosolic domain induced by Ca2+ binding and the conformational changes of the VSD induced by membrane voltage changes trigger the opening of the pore-gate domain. Although some structural information of these individual functional domains is available, how the interactions among these domains, especially the noncovalent interactions, control the dynamic gating process of BK channels is still not clear. Previous studies discovered that intracellular Mg2+ binds to an interdomain binding site consisting of D99 and N172 from the membrane-spanning domain and E374 and E399 from the cytosolic domain. The bound Mg2+ at this narrow interdomain interface activates the BK channel through an electrostatic interaction with a positively charged residue in the VSD. In this study, we investigated the potential interdomain interactions between the Mg2+-coordination residues and their effects on channel gating. By introducing different charges to these residues, we discovered a native interdomain interaction between D99 and E374 that can affect BK channel activation. To understand the underlying mechanism of the interdomain interactions between the Mg2+-coordination residues, we introduced artificial electrostatic interactions between residues 172 and 399 from two different domains. We found that the interdomain interactions between these two positions not only alter the local conformations near the Mg2+-binding site but also change distant conformations including the pore-gate domain, thereby affecting the voltage- and Ca2+-dependent activation of the BK channel. These results illustrate the importance of interdomain interactions to the allosteric gating mechanisms of BK channels.

scholarly journals Revealing the structural origin of the redox-Bohr effect: the first solution structure of a cytochrome from Geobacter sulfurreducens

2011 ◽  
Vol 441 (1) ◽  
pp. 179-187 ◽  
Author(s):  
Leonor Morgado ◽  
Vítor B. Paixão ◽  
Marianne Schiffer ◽  
P. Raj Pokkuluri ◽  
Marta Bruix ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Solution Structure ◽  
Structural Information ◽  
Atp Synthesis ◽  
Electronic Energy ◽  
Geobacter Sulfurreducens ◽  
Backbone Dynamics ◽  
Protonmotive Force ◽  
Functional Roles ◽  
Redox Partners

Gs (Geobacter sulfurreducens) can transfer electrons to the exterior of its cells, a property that makes it a preferential candidate for the development of biotechnological applications. Its genome encodes over 100 cytochromes and, despite their abundance and key functional roles, to date there is no structural information for these proteins in solution. The trihaem cytochrome PpcA might have a crucial role in the conversion of electronic energy into protonmotive force, a fundamental step for ATP synthesis in the presence of extracellular electron acceptors. In the present study, 15N-labelled PpcA was produced and NMR spectroscopy was used to determine its solution structure in the fully reduced state, its backbone dynamics and the pH-dependent conformational changes. The structure obtained is well defined, with an average pairwise rmsd (root mean square deviation) of 0.25 Å (1 Å=0.1 nm) for the backbone atoms and 0.99 Å for all heavy atoms, and constitutes the first solution structure of a Gs cytochrome. The redox-Bohr centre responsible for controlling the electron/proton transfer was identified, as well as the putative interacting regions between PpcA and its redox partners. The solution structure of PpcA will constitute the foundation for studies aimed at mapping out in detail these interacting regions.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

Rapid Elaboration of Fragments into Leads Applied to Bromodomain-3 Extra Terminal Domain

2020 ◽  
Author(s):  
Luke Adams ◽  
Lorna E. Wilkinson-White ◽  
Menachem J. Gunzburg ◽  
Stephen J. Headey ◽  
Martin J. Scanlon ◽  
...  
Keyword(s):  
Binding Site ◽  
Systematic Approach ◽  
Structural Information ◽  
Peptide Binding ◽  
Chemical Diversity ◽  
Chemical Probes ◽  
Protein Targets ◽  
Structure Activity ◽  
Peptide Binding Site ◽  
Selection Of

The development of low-affinity fragment hits into higher affinity leads is a major hurdle in fragment-based drug design. Here we demonstrate an approach for the Rapid Elaboration of Fragments into Leads (REFiL) applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure-activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. Upon completion of a fragment screen against Bromodomain-3 extra terminal (BRD3-ET) domain we applied the REFiL workflow, which allowed us to develop a series of tetrahydrocarbazole ligands that bind to the peptide binding site of BRD3-ET. With REFiL we were able to rapidly improve binding affinity >30-fold. The REFiL workflow can be applied readily to a broad range of protein targets without the need of a structure, allowing the efficient evolution of low-affinity fragments into higher affinity leads and chemical probes.<br>

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

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