The Ligand-Free State of the TPP Riboswitch: A Partially Folded RNA Structure

Riboswitches are elements of mRNA that regulate gene expression by undergoing structural changes upon binding of small ligands. Although the structures of several riboswitches have been solved with their ligands bound, the ligand-free states of only a few riboswitches have been characterized. The ligand-free state is as important for the functionality of the riboswitch as the ligand-bound form, but the ligand-free state is often a partially folded structure of the RNA, with conformational heterogeneity that makes it particularly challenging to study. Here, we present models of the ligand-free state of a thiamine pyrophosphate riboswitch that are derived from a combination of complementary experimental and computational modeling approaches. We obtain a global picture of the molecule using small-angle X-ray scattering data and use an RNA structure modeling software, MC-Sym, to fit local structural details to these data on an atomic scale. We have used two different approaches to obtaining these models. Our first approach develops a model of the RNA from the structures of its constituent junction fragments in isolation. The second approach treats the RNA as a single entity, without bias from the structure of its individual constituents. We find that both approaches give similar models for the ligand-free form, but the ligand-bound models differ for the two approaches, and only the models from the second approach agree with the ligand-bound structure known previously from X-ray crystallography. Our models provide a picture of the conformational changes that may occur in the riboswitch upon binding of its ligand. Our results also demonstrate the power of combining experimental small-angle X-ray scattering data with theoretical structure prediction tools in the determination of RNA structures beyond riboswitches.     

Influence of ground-state structure and Mg2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain

Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop-loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gsw(loop)) in the absence of Mg(2+). However, if Mg(2+) is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop-loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gsw(loop) is tunable through variation of the Mg(2+) concentration. We quantitatively describe the influence of distinct Mg(2+) concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution.     

Ligand-free and -bound structures of the binding protein (LivJ) of the Escherichia coli ABC leucine/isoleucine/valine transport system: trajectory and dynamics of the interdomain rotation and ligand specificity

The leucine/isoleucine/valine-binding protein (LIVBP or LivJ) serves as the primary high-affinity receptor of the Escherichia coli ABC-type transporter for the three aliphatic amino acids. The first structure of LIVBP determined previously without bound ligand showed a molecule comprised of two domains which are far apart and bisected by a wide open, solvent-accessible cleft. Here we report the crystal structures of another ligand-free state and three complexes with the aliphatic amino acids. In the present ligand-free structure, the two domains are farther apart. In the three very similar complex structures, the two domains are in close proximity, and each desolvated ligand is completely engulfed in the cleft and bound by both domains. The two different ligand-free structures, combined with those of the very similar ligand-bound structures, indicate the trajectory and backbone torsion angle changes of the hinges that accompany domain closure and play crucial functional roles. The amino acids are bound by polar and nonpolar interactions, occurring predominantly in one domain. Consistent with the protein specificity, the aliphatic side chains of the ligands lie in a hydrophobic pocket fully formed following domain or cleft closure. Comparison of the structures of LIVBP with several different binding proteins indicates no correlations between the magnitudes of the hinge-bending angles and the protein masses, the ligand sizes, or the number of segments connecting the two domains. Results of normal-mode analysis and molecular dynamics simulations are consistent with the trajectory and intrinsic flexibility of the interdomain hinges and the dominance of one domain in ligand binding in the open state.     

Conformational Changes and Ligand Recognition of Escherichia colid-Xylose Binding Protein Revealed

ATP binding cassette transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing cognate ligand and directing it to the appropriate membrane permease. Here we report the X-ray structures of d-xylose binding protein from Escherichia coli in ligand-free open form, ligand-bound open form, and ligand-bound closed form at 2.15 Å, 2.2 Å, and 2.2 Å resolutions, respectively. The ligand-bound open form is the first such structure to be reported at high resolution; the combination of the three different forms from the same protein furthermore gives unprecedented details concerning the conformational changes involved in binding protein function. As is typical of the structural family, the protein has two similar globular domains, which are connected by a three-stranded hinge region. The open liganded structure shows that xylose binds first to the C-terminal domain, with only very small conformational changes resulting. After a 34° closing motion, additional interactions are formed with the N-terminal domain; changes in this domain are larger and serve to make the structure more ordered near the ligand. An analysis of the interactions suggests why xylose is the preferred ligand. Furthermore, a comparison with the most closely related proteins in the structural family shows that the conformational changes are distinct in each type of binding protein, which may have implications for how the individual proteins act in concert with their respective membrane permeases.     

Variations in clique and community patterns in protein structures during allosteric communication: investigation of dynamically equilibrated structures of methionyl tRNA synthetase complexes

The allosteric concept has played a key role in understanding the biological functions of proteins. The rigidity or plasticity and the conformational population are the two important ideas invoked in explaining the allosteric effect. Although molecular insights have been gained from a large number of structures, a precise assessment of the ligand-induced conformational changes in proteins at different levels, ranging from gross topology to intricate details, remains a challenge. In this study, we have explored the conformational changes in the complexes of methionyl tRNA synthetase (MetRS) through novel network parameters such as cliques and communities, which identify the rigid regions in the protein structure networks (PSNs) constructed from the noncovalent interactions of amino acid side chains. MetRS belongs to the aminoacyl tRNA synthetase (aaRS) family that plays a crucial role in the translation of genetic code. These enzymes are modular with distinct domains from which extensive genetic, kinetic, and structural data are available, highlighting the role of interdomain communication. The network parameters evaluated here on the conformational ensembles of MetRS complexes, generated from molecular dynamics simulations, have enabled us to understand the interdomain communication in detail. Additionally, the characterization of conformational changes in terms of cliques and communities has also become possible, which had eluded conventional analyses. Furthermore, we find that most of the residues participating in cliques and communities are strikingly different from those that take part in long-range communication. The cliques and communities evaluated here for the first time on PSNs have beautifully captured the local geometries in detail within the framework of global topology. Here the allosteric effect is revealed at the residue level via identification of the important residues specific for structural rigidity and functional flexibility in MetRS. This ought to enhance our understanding of the functioning of aaRS in general.     

Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis.

The periplasmic maltodextrin binding protein of Escherichia coli serves as an initial receptor for the active transport of and chemotaxis toward maltooligosaccharides. The three-dimensional structure of the binding protein complexed with maltose has been previously reported [Spurlino, J. C., Lu, G.-Y., & Quiocho, F. A. (1991) J. Biol. Chem. 266, 5202-5219]. Here we report the structure of the unliganded form of the binding protein refined to 1.8-A resolution. This structure, combined with that for the liganded form, provides the first crystallographic evidence that a major ligand-induced conformational change occurs in a periplasmic binding protein. The unliganded structure shows a rigid-body "hinge-bending" between the two globular domains by approximately 35 degrees, relative to the maltose-bound structure, opening the sugar binding site groove located between the two domains. In addition, there is an 8 degrees twist of one domain relative to the other domain. The conformational changes observed between this structure and the maltose-bound structure are consistent with current models of maltose/maltodextrin transport and maltose chemotaxis and solidify a mechanism for receptor differentiation between the ligand-free and ligand-bound forms in signal transduction.     

Ligand-induced conformational changes in a thermophilic ribose-binding protein

Background

Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments.

Results

Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation ( ^app T _m value is 108°C) than the mesophilic Escherichia coli homolog (ecRBP) ( ^app T _m value is 56°C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved.

Conclusion

Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state.     

Ligand-induced melting reaction of specific-sequence DNA molecules

An algorithm for the calculation of ligand-induced helix–coil transitions of macromolecules of any specific sequence was derived. The probabilities of each residue to be in helix and ligand-free, helix and ligand-bound, coil and ligand-free, and coil and ligand-bound states can be calculated by extending the recursion relation formula proposed by D. Poland [(1974) Biopolymers 13 , 1859–1871]. Calculations using hypothetical DNAs having block heterogeneities for melting reactions showed that cooperative binding specific to single strands weakens the effect of the heterogeneity. Fine structures in the melting profiles or cooperatively melting regions, which have so far been found in thermal melting experiments, were predicted to exist in ligand-induced melting reactions for natural DNA fragments.     

残基相互作用网络特征与木聚糖酶耐热性的关系

残基相互作用网络是体现蛋白质中残基与残基之间协同和制约关系的重要形式。残基相互作用网络的拓扑性质以及社团结构与蛋白质的功能和性质有密切的关系。本文在构建一系列耐热木聚糖酶和常温木聚糖酶的残基相互作用网络后,通过计算网络的度、聚类系数、连接强度、特征路径长度、接近中心性、介数中心性等拓扑参数来确定网络拓扑结构与木聚糖酶耐热性的关系。识别残基相互作用网络的hub点,分析hub点的亲疏水性、带电性以及各种氨基酸在hub点中所占的比例。进一步使用GA-Net算法对网络进行社团划分,并计算社团的规模、直径和密度。网络的高平均度、高连接强度、以及更短的最短路径等表明耐热木聚糖酶残基相互作用网络的结构更加紧密;耐热木聚糖酶网络中的hub节点比常温木聚糖酶网络hub节点具有更多的疏水性残基,hub点中Phe、Ile、Val的占比更高。社团检测后发现,耐热木聚糖酶网络拥有更大的社团规模、较小的社团直径和较大的社团密度。社团规模越大表明耐热木聚糖酶的氨基酸残基更倾向于形成大的社团,而较小的社团直径和较大的社团密度则表明社团内部氨基酸残基的相互作用比常温木聚糖酶更强。     

Evidence of Conformational Selection Driving the Formation of Ligand Binding Sites in Protein-Protein Interfaces

Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. This agrees with the conformational selection model of molecular recognition, which assumes such pre-existing conformations. The analysis also shows the maximum level of similarity between unbound and bound states that is achieved without any influence from a ligand. Further shift toward the bound structure assumes protein-peptide or protein-ligand interactions, either selecting higher energy conformations that are not part of the NMR ensemble, or leading to induced fit. Thus, forming the sites in protein-protein interfaces that bind peptides and can be targeted by small ligands always includes conformational selection, although other recognition mechanisms may also be involved.     

A network representation of protein structures: implications for protein stability

This study views each protein structure as a network of noncovalent connections between amino acid side chains. Each amino acid in a protein structure is a node, and the strength of the noncovalent interactions between two amino acids is evaluated for edge determination. The protein structure graphs (PSGs) for 232 proteins have been constructed as a function of the cutoff of the amino acid interaction strength at a few carefully chosen values. Analysis of such PSGs constructed on the basis of edge weights has shown the following: 1), The PSGs exhibit a complex topological network behavior, which is dependent on the interaction cutoff chosen for PSG construction. 2), A transition is observed at a critical interaction cutoff, in all the proteins, as monitored by the size of the largest cluster (giant component) in the graph. Amazingly, this transition occurs within a narrow range of interaction cutoff for all the proteins, irrespective of the size or the fold topology. And 3), the amino acid preferences to be highly connected (hub frequency) have been evaluated as a function of the interaction cutoff. We observe that the aromatic residues along with arginine, histidine, and methionine act as strong hubs at high interaction cutoffs, whereas the hydrophobic leucine and isoleucine residues get added to these hubs at low interaction cutoffs, forming weak hubs. The hubs identified are found to play a role in bringing together different secondary structural elements in the tertiary structure of the proteins. They are also found to contribute to the additional stability of the thermophilic proteins when compared to their mesophilic counterparts and hence could be crucial for the folding and stability of the unique three-dimensional structure of proteins. Based on these results, we also predict a few residues in the thermophilic and mesophilic proteins that can be mutated to alter their thermal stability.     

残基相互作用网络特征与木聚糖酶耐热性的关系

残基相互作用网络是体现蛋白质中残基与残基之间协同和制约关系的重要形式。残基相互作用网络的拓扑性质以及社团结构与蛋白质的功能和性质有密切的关系。本文在构建一系列耐热木聚糖酶和常温木聚糖酶的残基相互作用网络后,通过计算网络的度、聚类系数、连接强度、特征路径长度、接近中心性、介数中心性等拓扑参数来确定网络拓扑结构与木聚糖酶耐热性的关系。识别残基相互作用网络的hub点,分析hub点的亲疏水性、带电性以及各种氨基酸在hub点中所占的比例。进一步使用GA-Net算法对网络进行社团划分,并计算社团的规模、直径和密度。网络的高平均度、高连接强度、以及更短的最短路径等表明耐热木聚糖酶残基相互作用网络的结构更加紧密;耐热木聚糖酶网络中的hub节点比常温木聚糖酶网络hub节点具有更多的疏水性残基,hub点中Phe、Ile、Val的占比更高。社团检测后发现,耐热木聚糖酶网络拥有更大的社团规模、较小的社团直径和较大的社团密度。社团规模越大表明耐热木聚糖酶的氨基酸残基更倾向于形成大的社团,而较小的社团直径和较大的社团密度则表明社团内部氨基酸残基的相互作用比常温木聚糖酶更强。     

Mechanisms for ligand binding to GluR0 ion channels: crystal structures of the glutamate and serine complexes and a closed apo state

High-resolution structures of the ligand binding core of GluR0, a glutamate receptor ion channel from Synechocystis PCC 6803, have been solved by X-ray diffraction. The GluR0 structures reveal homology with bacterial periplasmic binding proteins and the rat GluR2 AMPA subtype neurotransmitter receptor. The ligand binding site is formed by a cleft between two globular alpha/beta domains. L-Glutamate binds in an extended conformation, similar to that observed for glutamine binding protein (GlnBP). However, the L-glutamate gamma-carboxyl group interacts exclusively with Asn51 in domain 1, different from the interactions of ligand with domain 2 residues observed for GluR2 and GlnBP. To address how neutral amino acids activate GluR0 gating we solved the structure of the binding site complex with L-serine. This revealed solvent molecules acting as surrogate ligand atoms, such that the serine OH group makes solvent-mediated hydrogen bonds with Asn51. The structure of a ligand-free, closed-cleft conformation revealed an extensive hydrogen bond network mediated by solvent molecules. Equilibrium centrifugation analysis revealed dimerization of the GluR0 ligand binding core with a dissociation constant of 0.8 microM. In the crystal, a symmetrical dimer involving residues in domain 1 occurs along a crystallographic 2-fold axis and suggests that tetrameric glutamate receptor ion channels are assembled from dimers of dimers. We propose that ligand-induced conformational changes cause the ion channel to open as a result of an increase in domain 2 separation relative to the dimer interface.     

Effects of ligand binding on dynamics of fatty acid binding protein and interactions with membranes

Intracellular transport of fatty acids involves binding of ligands to their carrier fatty acid binding proteins (FABPs) and interactions of ligand-free and -bound FABPs with membranes. Previous studies focused on ligand-free FABPs. Here, our amide hydrogen exchange data showed that oleic acid binding to human intestinal FABP (hIFABP) stabilizes the protein, most likely through enhancing the hydrogen-bonding network, and induces rearrangement of sidechains even far away from the ligand binding site. Using NMR relaxation techniques, we found that the ligand binding affects not only conformational exchanges between major and minor states but also the affinity of hIFABP to nanodiscs. Analyses of the relaxation and amide exchange data suggested that two minor native-like states existing in both ligand-free and -bound hIFABPs originate from global “breathing” motions, while one minor native-like state comes from local motions. The amide hydrogen exchange data also indicated that helix αII undergoes local unfolding through which ligands can exit from the binding cavity.     

Validation of protein structure models using network similarity score

Accurate structural validation of proteins is of extreme importance in studies like protein structure prediction, analysis of molecular dynamic simulation trajectories and finding subtle changes in very similar structures. The benchmarks for today's structure validation are scoring methods like global distance test‐total structure (GDT‐TS), TM‐score and root mean square deviations (RMSD). However, there is a lack of methods that look at both the protein backbone and side‐chain structures at the global connectivity level and provide information about the differences in connectivity. To address this gap, a graph spectral based method (NSS—network similarity score) which has been recently developed to rigorously compare networks in diverse fields, is adopted to compare protein structures both at the backbone and at the side‐chain noncovalent connectivity levels. In this study, we validate the performance of NSS by investigating protein structures from X‐ray structures, modeling (including CASP models), and molecular dynamics simulations. Further, we systematically identify the local and the global regions of the structures contributing to the difference in NSS, through the components of the score, a feature unique to this spectral based scoring scheme. It is demonstrated that the method can quantify subtle differences in connectivity compared to a reference protein structure and can form a robust basis for protein structure comparison. Additionally, we have also introduced a network‐based method to analyze fluctuations in side chain interactions (edge‐weights) in an ensemble of structures, which can be an useful tool for the analysis of MD trajectories.     

Structural coupling between FKBP12 and buried water

Globular proteins often contain structurally well-resolved internal water molecules. Previously, we reported results from a molecular dynamics study that suggested that buried water (Wat3) may play a role in modulating the structure of the FK506 binding protein-12 (FKBP12) (Park and Saven, Proteins 2005; 60:450-463). In particular, simulations suggested that disrupting a hydrogen bond to Wat3 by mutating E60 to either A or Q would cause a structural perturbation involving the distant W59 side chain, which rotates to a new conformation in response to the mutation. This effectively remodels the ligand-binding pocket, as the side chain in the new conformation is likely to clash with bound FK506. To test whether the protein structure is in effect modulated by the binding of a buried water in the distance, we determined high-resolution (0.92-1.29 A) structures of wild-type FKBP12 and its two mutants (E60A, E60Q) by X-ray crystallography. The structures of mutant FKBP12 show that the ligand-binding pocket is indeed remodeled as predicted by the substitution at position 60, even though the water molecule does not directly interact with any of the amino acids of the binding pocket. Thus, these structures support the view that buried water molecules constitute an integral, noncovalent component of the protein structure. Additionally, this study provides an example in which predictions from molecular dynamics simulations are experimentally validated with atomic precision, thus showing that the structural features of protein-water interactions can be reliably modeled at a molecular level.     

Intermolecular β-Strand Networks Avoid Hub Residues and Favor Low Interconnectedness: A Potential Protection Mechanism against Chain Dissociation upon Mutation

Altogether few protein oligomers undergo a conformational transition to a state that impairs their function and leads to diseases. But when it happens, the consequences are not harmless and the so-called conformational diseases pose serious public health problems. Notorious examples are the Alzheimer''s disease and some cancers associated with a conformational change of the amyloid precursor protein (APP) and of the p53 tumor suppressor, respectively. The transition is linked with the propensity of β-strands to aggregate into amyloid fibers. Nevertheless, a huge number of protein oligomers associate chains via β-strand interactions (intermolecular β-strand interface) without ever evolving into fibers. We analyzed the layout of 1048 intermolecular β-strand interfaces looking for features that could provide the β-strands resistance to conformational transitions. The interfaces were reconstructed as networks with the residues as the nodes and the interactions between residues as the links. The networks followed an exponential decay degree distribution, implying an absence of hubs and nodes with few links. Such layout provides robustness to changes. Few links per nodes do not restrict the choices of amino acids capable of making an interface and maintain high sequence plasticity. Few links reduce the “bonding” cost of making an interface. Finally, few links moderate the vulnerability to amino acid mutation because it entails limited communication between the nodes. This confines the effects of a mutation to few residues instead of propagating them to many residues via hubs. We propose that intermolecular β-strand interfaces are organized in networks that tolerate amino acid mutation to avoid chain dissociation, the first step towards fiber formation. This is tested by looking at the intermolecular β-strand network of the p53 tetramer.