Protein Domain Motions Research Articles

Database of ligand-induced domain movements in enzymes.

BackgroundConformational change induced by the binding of a substrate or coenzyme is a poorly understood stage in the process of enzyme catalysed reactions. For enzymes that exhibit a domain movement, the conformational change can be clearly characterized and therefore the opportunity exists to gain an understanding of the mechanisms involved. The development of the non-redundant database of protein domain movements contains examples of ligand-induced domain movements in enzymes, but this valuable data has remained unexploited.DescriptionThe domain movements in the non-redundant database of protein domain movements are those found by applying the DynDom program to pairs of crystallographic structures contained in Protein Data Bank files. For each pair of structures cross-checking ligands in their Protein Data Bank files with the KEGG-LIGAND database and using methods that search for ligands that contact the enzyme in one conformation but not the other, the non-redundant database of protein domain movements was refined down to a set of 203 enzymes where a domain movement is apparently triggered by the binding of a functional ligand. For these cases, ligand binding information, including hydrogen bonds and salt-bridges between the ligand and specific residues on the enzyme is presented in the context of dynamical information such as the regions that form the dynamic domains, the hinge bending residues, and the hinge axes.ConclusionThe presentation at a single website of data on interactions between a ligand and specific residues on the enzyme alongside data on the movement that these interactions induce, should lead to new insights into the mechanisms of these enzymes in particular, and help in trying to understand the general process of ligand-induced domain closure in enzymes. The website can be found at:

Crystal structure of human adenylate kinase 4 (L171P) suggests the role of hinge region in protein domain motion

It is well known that motion of LID and NMP-binding (NMP bind) domains in adenylate kinase (AK) is important in ligand binding and catalysis. However, the nature of such domain motions is poorly characterized. One of the critical hinge regions is hinge IV, which connects the CORE and LID domains. In addition, the hinge IV contains a strictly conserved residue, L171, in the AK family. To investigate the role of hinge IV, crystal structure of human adenylate kinase 4 (AK4) L171P mutant was determined. This mutation dramatically changes the orientation of the LID domain, which could be described as a novel twisted-and-closed conformation in contrast to the open and closed conformations in other AKs. This mutant provides a new example of domain motions in AK family.

Using affinity chromatography to engineer and characterize pH‐dependent protein switches

Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione-S-transferase to convert this protein into a pH-dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH-dependent GSH-binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector-specific allostery into a protein structure.

The Atomistic Mechanism of Conformational Transition in Adenylate Kinase: A TEE-REX Molecular Dynamics Study

We report on an atomistic molecular dynamics simulation of the complete conformational transition of Escherichia coli adenylate kinase (ADK) using the recently developed TEE-REX algorithm. Two phases characterize the transition pathway of ADK, which folds into the domains CORE and LID and the AMP binding domain AMPbd. Starting from the closed conformation, half-opening of the AMPbd precedes a partially correlated opening of the LID and AMPbd, defining the second phase. A highly stable salt bridge D118-K136 at the LID-CORE interface, contributing substantially to the total nonbonded LID-CORE interactions, was identified as a major factor that stabilizes the open conformation. Alternative transition pathways, such as AMPbd opening following LID opening, seem unlikely, as full transition events were not observed along this pathway. The simulation data indicate a high enthalpic penalty, possibly obstructing transitions along this route.

Motor step size and ATP coupling efficiency of the dsDNA translocase EcoR124I

The Type I restriction-modification enzyme EcoR124I is an archetypical helicase-based dsDNA translocase that moves unidirectionally along the 3′–5′ strand of intact duplex DNA. Using a combination of ensemble and single-molecule measurements, we provide estimates of two physicochemical constants that are fundamental to a full description of motor protein activity—the ATP coupling efficiency (the number of ATP consumed per base pair) and the step size (the number of base pairs transported per motor step). Our data indicate that EcoR124I makes small steps along the DNA of 1 bp in length with 1 ATP consumed per step, but with some uncoupling of the ATPase and translocase cycles occurring so that the average number of ATP consumed per base pair slightly exceeds unity. Our observations form a framework for understanding energy coupling in a great many other motors that translocate along dsDNA rather than ssDNA.

1P-051 分子動力学計算によるタンパク質のドメイン運動に関する研究(蛋白質・構造機能相関(1),第46回日本生物物理学会年会)

1P-051 分子動力学計算によるタンパク質のドメイン運動に関する研究(蛋白質・構造機能相関(1),第46回日本生物物理学会年会)

What is the relationship between the global structures of apo and holo proteins?

It is well known that ligand binding and release may induce a wide range of structural changes in a receptor protein, varying from small movements of loops or side chains in the binding pocket to large-scale domain hinge-bending and shear motions or even partial unfolding that facilitates the capture and release of a ligand. An interesting question is what in general are the conformational changes triggered by ligand binding? The aim of this work is analyze the magnitude of structural changes in a protein resulting from ligand binding to assess if the state of ligand binding needs to be included in template-based protein structure prediction algorithms. To address this issue, a nonredundant dataset of 521 paired protein structures in the ligand-free and ligand-bound form was created and used to estimate the degree of both local and global structure similarity between the apo and holo forms. In most cases, the proteins undergo relatively small conformational rearrangements of their tertiary structure upon ligand binding/release (most root-mean-square-deviations from native, RMSD, are <1 A). However, a clear difference was observed between single- and multiple-domain proteins. For the latter, RMSD changes greater than 1 A and sometimes larger were found for almost 1/3 of the cases; these are mainly associated with large-scale hinge-bending movements of entire domains. The changes in the mutual orientation of individual domains in multiple-domain proteins upon ligand binding were investigated using a mechanistic model based on mass-weighted principal axes as well as interface buried surface calculations. Some preferences toward the anticipated mechanism of protein domain movements are predictable based on the examination of just the ligand-free structural form. These results have applications to protein structure prediction, particularly in the context of protein domain assembly, if additional information concerning ligand binding is exploited.

Docking of ATP to Ca‐ATPase: Considering Protein Domain Motions.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Domain–domain motions in proteins from time-modulated pseudocontact shifts

In recent years paramagnetic NMR derived structural constraints have become increasingly popular for the study of biomolecules. Some of these are based on the distance and angular dependences of pseudo contact shifts (PCSs). When modulated by internal motions PCSs also become sensitive reporters on molecular dynamics. We present here an investigation of the domain-domain motion in a two domain protein (PA0128) through time-modulation of PCSs. PA0128 is a protein of unknown function from Pseudomonas aeruginosa (PA) and contains a Zn(2+) binding site in the N-terminal domain. When substituted with Co(2+) in the binding site, several resonances from the C-terminal domain showed severe line broadening along the (15)N dimension. Relaxation compensated CPMG experiments revealed that the dramatic increase in the (15)N linewidth came from contributions of chemical exchange. Since several sites with perturbed relaxation are localized to a single beta-strand region, and since extracted timescales of motion for the perturbed sites are identical, and since the magnitude of the chemical exchange contributions is consistent with PCSs, the observed rate enhancements are interpreted as the result of concerted domain motion on the timescale of a few milliseconds. Given the predictability of PCS differences and the easy interpretation of the experimental results, we suggest that these effects might be useful in the study of molecular processes occurring on the millisecond to microsecond timescale.

Docking of ATP to Ca-ATPase: Considering Protein Domain Motions

Most standard molecular docking algorithms take into account only ligand flexibility, while numerous studies demonstrate that receptor flexibility may be also important. While some efficient methods have been proposed to take into account local flexibility of protein side chains, the influence of large-scale domain motions on the docking results still represents a challenge for computational methods. In this work we compared the results of ATP docking to different models of Ca-ATPase: crystallographic apo- and holo-forms of the enzyme as well as "flexible" target models generated via molecular dynamics (MD) simulations in water. MD simulations were performed for two different apo-forms and one holo-form of Ca2+-ATPase and reveal large-scale domain motions of type "closure", which is consistent with experimental structures. Docking to a set of MD-conformers yielded correct solutions with ATP bound in both domains regardless of the starting Ca2+-ATPase structure. Also, special attention was paid to proper ranking of docking solutions and some particular features of different scoring functions and their applicability for the model of "flexible" receptor. Particularly, the results of docking ATP were ranked by a scoring criterion specially designed to estimate ATP-protein interactions. This criterion includes stacking and hydrophobic interactions characteristic of ATP-protein complexes. The performance of this ligand-specific scoring function was considerably better than that of a standard scoring function used in the docking algorithm.

Last Stop on the Road to Repair: Structure of E. coli DNA Ligase Bound to Nicked DNA-Adenylate

NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria and essential for growth. Their distinctive substrate specificity and domain organization vis-a-vis human ATP-dependent ligases make them outstanding targets for anti-infective drug discovery. We report here the 2.3 A crystal structure of Escherichia coli LigA bound to an adenylylated nick, which captures LigA in a state poised for strand closure and reveals the basis for nick recognition. LigA envelopes the DNA within a protein clamp. Large protein domain movements and remodeling of the active site orchestrate progression through the three chemical steps of the ligation reaction. The structure inspires a strategy for inhibitor design.

2P061 分子動力学計算によるタンパク質のドメイン運動に関する研究(蛋白質(構造・構造機能相関),ポスター発表,第45回日本生物物理学会年会)

2P061 分子動力学計算によるタンパク質のドメイン運動に関する研究(蛋白質(構造・構造機能相関),ポスター発表,第45回日本生物物理学会年会)

Coupled protein domain motion inTaqpolymerase revealed by neutron spin-echo spectroscopy

Long-range conformational changes in proteins are ubiquitous in biology for the transmission and amplification of signals; such conformational changes can be triggered by small-amplitude, nanosecond protein domain motion. Understanding how conformational changes are initiated requires the characterization of protein domain motion on these timescales and on length scales comparable to protein dimensions. Using neutron spin-echo spectroscopy (NSE), normal mode analysis, and a statistical-mechanical framework, we reveal overdamped, coupled domain motion within DNA polymerase I from Thermus aquaticus (Taq polymerase). This protein utilizes correlated domain dynamics over 70 angstroms to coordinate nucleotide synthesis and cleavage during DNA synthesis and repair. We show that NSE spectroscopy can determine the domain mobility tensor, which determines the degree of dynamical coupling between domains. The mobility tensor defines the domain velocity response to a force applied to it or to another domain, just as the sails of a sailboat determine its velocity given the applied wind force. The NSE results provide insights into the nature of protein domain motion that are not appreciated by conventional biophysical techniques.

A comprehensive and non-redundant database of protein domain movements

The current DynDom database of protein domain motions is a user-created database that suffers from selectivity and redundancy. The aim of the analysis presented here was to overcome both these limitations and to produce both a comprehensive and a non-redundant description of domain movements from structures stored in the current protein data bank. A multi-step procedure is applied that starts with grouping proteins in the structural databank into families based on sequence similarity. Multiple sequence alignment, conformational clustering and a dimensional clustering method based on the Gram-Schmidt algorithm are applied to members of each family to remove dynamic redundancy in their domain movements. Representative domain movements are described in terms of domains, hinge axes and hinge-bending residues using the DynDom program. The results show that within an average family of 11.5 members, there are on average only 1.31 different domain movements indicating a high redundancy in the movements these structures represent. This verifies earlier findings that domain movements are usually highly controlled. Despite the removal of this considerable redundancy, the process has resulted in double the number of domain movements stored in the user-created database. The data are organized in a relational database with a web-interface. The database can be browsed and searched at http://www.cmp.uea.ac.uk/dyndom sjh@cmp.uea.ac.uk.

Trehalose-enzyme interactions result in structure stabilization and activity inhibition. The role of viscosity.

Stress resistance is essential for survival. The mechanisms of molecule stabilization during stress are of interest for biotechnology, where many enzymes and other biomolecules are increasingly used at high temperatures and/or salt concentrations. Diverse organisms, exhibit rapid synthesis and accumulation of the disaccharide trehalose in response to stress. Trehalose is also rapidly hydrolyzed as soon as stress ends. In isolated enzymes, trehalose stabilizes both, structure and activity. In contrast, at optimal assay conditions, trehalose inhibits enzyme activity. A general mechanism underlying the trehalose effects observed at all temperatures probably is the trehalose-mediated increase in solution viscosity that leads to protein domain motion inhibition. This may be analyzed using Kramer's theory. The role of viscosity in the effects of trehalose is analyzed in examples from the literature and in studies on the plasma membrane H(+)-ATPase from Kluyveromyces lactis. In the cell, it may be proposed that the large concentration of trehalose reached during stress stabilizes structures through viscosity. However, once stress ends trehalose has to be rapidly hydrolyzed in order to avoid the viscosity-mediated inhibition of enzymes.

Locking the DNA topoisomerase I protein clamp inhibits DNA rotation and induces cell lethality.

Eukaryotic DNA topoisomerase I (Top1) is a monomeric protein clamp that functions in DNA replication, transcription, and recombination. Opposable "lip" domains form a salt bridge to complete Top1 protein clamping of duplex DNA. Changes in DNA topology are catalyzed by the formation of a transient phosphotyrosyl linkage between the active-site Tyr-723 and a single DNA strand. Substantial protein domain movements are required for DNA binding, whereas the tight packing of DNA within the covalent Top1-DNA complex necessitates some DNA distortion to allow rotation. To investigate the effects of Top1-clamp closure on enzyme catalysis, molecular modeling was used to design a disulfide bond between residues Gly-365 and Ser-534, to crosslink protein loops more proximal to the active-site tyrosine than the protein loops held by the Lys-369-Glu-497 salt bridge. In reducing environments, Top1-Clamp was catalytically active. However, contrary to crosslinking the salt-bridge loops [Carey, J. F., Schultz, S. J., Sission, L., Fazzio, T. G. & Champoux, J. J. (2003) Proc. Natl. Acad. Sci. USA 100, 5640-5645], crosslinking the active-site proximal loops inhibited DNA rotation. Apparently, subtle alterations in Top1 clamp flexibility impact enzyme catalysis in vitro. Yet, the catalytically active Top1-Clamp was cytotoxic, even in the reducing environment of yeast cells. Remarkably, a shift in redox potential in glr1Delta cells converted the catalytically inactive Top1Y723F mutant clamp into a cellular toxin, which failed to induce an S-phase terminal phenotype. This cytotoxic mechanism is distinct from that of camptothecin chemotherapeutics, which stabilize covalent Top1-DNA complexes, and it suggests that the development of novel therapeutics that promote Top1-clamp closure is possible.

The DynDom database of protein domain motions.

A relational database has been developed based on the results from the application of the DynDom program to a number of proteins for which multiple X-ray conformers are available. The database is populated via a web-based tool that allows visitors to the website to run the DynDom program server-side by selecting pairs of X-ray conformers by Protein Data Bank code and chain identifier. The website can be found at: http://www.sys.uea.ac.uk/dyndom.

Correlation between normal modes in the 20–200 cm −1 frequency range and localized torsion motions related to certain collective motions in proteins

In certain biologically relevant collective motions, such as protein domain motions and sub-domain motions, large amplitude movements are localized in one or a few flexible regions consisting of a small number of residues. This paper explores the possible use of normal mode analysis in probing localized vibrational torsion motions in these flexible regions that may be related to certain collective motions. The normal modes of 10 structures of five proteins in different conformation (TRP repressor, calmodulin, calbindin D 9k, HIV-1 protease and troponin C), known to have shear or hinge domain or sub-domain motion, respectively, are analyzed. Our study identifies, for each structure, unique normal modes in the 20–200 cm −1 frequency range, whose corresponding motions are primarily concentrated in the region where large amplitude torsion movements of a known domain or sub-domain motion occur. This suggests possible correlation between normal modes at 20–200 cm −1 frequency range and initial fluctuational motions leading to localized collective motions in proteins, and thus the potential application of normal mode analysis in facilitating the study of biologically important localized motions in biomolecules.

Improvements in the analysis of domain motions in proteins from conformational change: DynDom version 1.50

DynDom is a program that analyses conformational change in proteins for dynamic domains, hinge axes, and hinge-bending regions. Here, a number of improvements and additions are reported which have been implemented in the new version 1.50. The most significant improvement is in the determination of the hinge-bending residues. A new routine also compares quantities relating to the main-chain dihedrals of bending residues with the hinge-bending motion. This version of the program can now be run from the DynDom website at: http://www.sys.uea.ac.uk/dyndom.

Mechanics of biomolecules

Over the last few years molecular biomechanics has emerged as a new field in which theoretical and experimental studies of the mechanics of proteins and nucleic acids have become a focus, and the importance of mechanical forces and motions to the fundamentals of biology and biochemistry has begun to be recognized. In particular, single-molecule biomechanics of DNA extension, bending and twisting; protein domain motion, deformation and unfolding; and the generation of mechanical forces and motions by biomolecular motors has become a new frontier in life sciences. There is an increasing need for a more systematic study of the basic issues involved in molecular biomechanics, and a more active participation of researchers in applied mechanics. Here we review some of the advances in this field over the last few years, explore the connection between mechanics and biochemistry, and discuss the concepts, issues, approaches and challenges, aiming to stimulating a broader interest in developing molecular biomechanics.