Using Rigidity Analysis to Probe Mutation-Induced Structural Changes in Proteins

Predicting the effect of a single amino acid substitution on the stability of a protein structure is a fundamental task in macromolecular modeling. It has relevance to drug design and understanding of disease-causing protein variants. We present KINARI-Mutagen, a web server for performing in silico mutation experiments on protein structures from the Protein Data Bank. Our rigidity-theoretical approach permits fast evaluation of the effects of mutations that may not be easy to perform in vitro, because it is not always possible to express a protein with a specific amino acid substitution. We use KINARI-Mutagen to identify critical residues, and we show that our predictions correlate with destabilizing mutations to glycine. In two in-depth case studies we show that the mutated residues identified by KINARI-Mutagen as critical correlate with experimental data, and would not have been identified by other methods such as Solvent Accessible Surface Area measurements or residue ranking by contributions to stabilizing interactions. We also generate 48 mutants for 14 proteins, and compare our rigidity-based results against experimental mutation stability data. KINARI-Mutagen is available at http://kinari.cs.umass.edu. © 2012 Imperial College Press

Allo-network drugs: Extension of the allosteric drug concept to protein-protein interaction and signaling networks

Allosteric drugs are usually more specific and have fewer side effects than orthosteric drugs targeting the same protein. Here, we overview the current knowledge on allosteric signal transmission from the network point of view, and show that most intra-protein conformational changes may be dynamically transmitted across protein-protein interaction and signaling networks of the cell. Allo-network drugs influence the pharmacological target protein indirectly using specific inter-protein network pathways. We show that allo-network drugs may have a higher efficiency to change the networks of human cells than those of other organisms, and can be designed to have specific effects on cells in a diseased state. Finally, we summarize possible methods to identify allo-network drug targets and sites, which may develop to a promising new area of systems-based drug design

Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT

Influence of Subunit Interface Mutations on Kinetic and Dynamic Properties of Alkaline Phosphatase from E. coli

Mutations, replacing amino acids involved in the formation of hydrogen bonds between subunits of dimeric alkaline phosphatase, have been introduced. Influence of mutations on kinetic properties and structural stability of mutant enzymes was established. In addition, alterations in protein dynamic properties have been studied using room temperature phosphorescence. Kinetic properties of both mutant enzymes were virtually the same, differing from the wild type enzyme in the kcat value that was almost twice lower. Changes in protein dynamic properties of mutant proteins, compared to the wild type enzyme, did not parallel changes in kinetic properties suggesting that an alteration in the rigidity of the Trp109 environment is not responsible for the reduction of kinetic properties. Instead, combined kinetic and dynamic consequences of introduced mutations suggest that breaking of specific links, involved in transmission of conformational change, could be responsible for altered kinetic properties. (doi: 10.5562/cca2168

Histidine substitution in the most flexible fragments of firefly luciferase modifies its thermal stability.

Molecular dynamics (MD) at two temperatures of 300 and 340 K identified two histidine residues, His461 and His489, in the most flexible regions of firefly luciferase, a light emitting enzyme. We therefore designed four protein mutants H461D, H489K, H489D and H489M to investigate their enzyme kinetic and thermodynamic stability changes. Substitution of His461 by aspartate (H461D) decreased ATP binding affinity, reduced the melting temperature of protein by around 25 degrees C and shifted its optimum temperature of activity to 10 degrees C. In line with the common feature of psychrophilic enzymes, the MD data showed that the overall flexibility of H461D was relatively high at low temperature, probably due to a decrease in the number of salt bridges around the mutation site. On the other hand, substitution of His489 by aspartate (H489D) introduced a new salt bridge between the C-terminal and N-terminal domains and increased protein rigidity but only slightly improved its thermal stability. Similar changes were observed for H489K and, to a lesser degree, H489M mutations. Based on our results we conclude that the MD simulation-based rational substitution of histidines by salt-bridge forming residues can modulate conformational dynamics in luciferase and shift its optimal temperature activity

Mechanistic behaviour and molecular interactions of heat shock protein 47 (HSP47)

This project involves the study of heat shock protein 47 (HSP47), which is a molecular chaperone crucial for collagen biosynthesis. It exhibits a high degree of sequence homology with members of the serine protease inhibitor (serpin) superfamily, though HSP47 does not possess the inhibitory activity. It is a single-substrate chaperone, and binds only to collagen. ‘Knock-out’ of the hsp47 gene impairs the secretion of correctly folded collagen triple helix molecules leading to embryonic lethality in mice. Thus the aim of this project was to elucidate the specific mechanism that governs the binding to and release from collagen at the molecular level, known as the ‘pH-switch mechanism’. Emphasis is given on histidine (His) residues as the HSP47-collagen dissociation pH is similar to the pKa of the imidazole side chain of His residues. Site directed mutagenesis was used to mutate surface His residues, based on a mouse HSP47 homology model. The effects of the mutations on the behaviour of HSP47 were then assessed by collagen binding assays and structural analyses with circular dichroism (CD). All mutants were found to have good solubility and retain their binding ability to collagen like wild-type HSP47 in batch assay, but perturbed behaviour was seen in column experiment. Mutation of His residue at position 191 (H191) causes the shift in the collagen dissociation pH, while mutation of H197 and/or 198 disrupt the specific HSP47-collagen interaction. H191, 197 and 198 are predicted to be located in the region near the C-terminus of strand 3 of β-sheet A (s3A) in the homology model, a region specifically known as the ‘breach cluster’ in serpin nomenclature. The extent of conformational rearrangement of this region was further investigated by means of intrinsic tryptophan fluorescence spectroscopy using a series of single tryptophan (Trp) mutants. Results from analyses performed on the mutants did not contradict the observation seen in His mutational work, as Trp residues in the ‘breach’ cluster are likely to be located in the dynamic region of HSP47 pH-triggered conformational change. In conclusion, this study establishes the importance of His residues in the ‘breach cluster’ to HSP47 pH-switch behaviour. Finally, a model for HSP47 pH-switch mechanism was proposed from data obtained via mutagenesis experiments. The model is hoped to assist future research into HSP47 cellular behaviour and will also be of great use in therapeutic applications involving the molecular chaperone

A conservation and rigidity based method for detecting critical protein residues

Background Certain amino acids in proteins play a critical role in determining their structural stability and function. Examples include flexible regions such as hinges which allow domain motion, and highly conserved residues on functional interfaces which allow interactions with other proteins. Detecting these regions can aid in the analysis and simulation of protein rigidity and conformational changes, and helps characterizing protein binding and docking. We present an analysis of critical residues in proteins using a combination of two complementary techniques. One method performs in-silico mutations and analyzes the protein\u27s rigidity to infer the role of a point substitution to Glycine or Alanine. The other method uses evolutionary conservation to find functional interfaces in proteins. Results We applied the two methods to a dataset of proteins, including biomolecules with experimentally known critical residues as determined by the free energy of unfolding. Our results show that the combination of the two methods can detect the vast majority of critical residues in tested proteins. Conclusions Our results show that the combination of the two methods has the potential to detect more information than each method separately. Future work will provide a confidence level for the criticalness of a residue to improve the accuracy of our method and eliminate false positives. Once the combined methods are integrated into one scoring function, it can be applied to other domains such as estimating functional interfaces

Measuring mechanical tension across the focal adhesion protein talin-1

Cell adhesion is an essential mechanism involved in many cellular processes, such as migration, proliferation and differentiation. The mechanical linkage between extracellular matrix and the f-actin cytoskeleton is mediated in specialized protein complexes, called focal adhesions. Key components of these cellular compartments are members of the integrin protein family; transmembrane proteins that connect to ligands in the extracellular matrix and recruit intracellular focal adhesion proteins. However, integrins have no catalytic function and cannot bind cytoskeletal components. The association with f-actin is mediated by intracellular adaptor molecules that link integrin tails and the cytoskeleton. These adhesion complexes not only mediate association with the extracellular matrix, but also serve as the mechanosensitive units of the cell. It has been known for some time that mechanical stimuli – as for example tissue rigidity – are epigenetic factors, regulating processes like organ development and stem cell differentiation. However, even though single components of the adhesion complex have been demonstrated to be involved in mechanosensitive processes, central mechanisms in mechanosensing through focal adhesions remained unknown. One of the major components responsible for the integrin-f-actin connection is the focal adhesion protein talin-1. Talin-1 directly binds intracellular integrin tails but also carries three f-actin binding sites and thus directly mediates the connection between extracellular Matrix and the cytoskeleton. Besides ist important role as integrin activator – and thus important promotor of integrin mediated adhesion – talin-1 has long been suspected as a mechanosensitive component in focal adhesions. Still, evidence of a regulatory role of talin-1 in mechanosensing in adhesive cells is still missing due to the lack of appropriate techniques. Using two single-molecule-calibrated FRET (Förster resonance energy transfer) based tension sensors, it could be demonstrated in this work that talin-1 is indeed subject to low-piconewton (pN) forces in integrin mediated adhesion processes. When localized in focal adhesion talin-1 bears forces of 7-10 pN. Regulation of talin-1 forces occurs through association with f-actin, either direct or indirect via binding of vinculin, a talin-1 interactor that strengthens the connection of talin-1 with the actin cytoskeleton. Disturbing the mechanical linkage of integrins to f-actin via talin-1 does not prevent integrin activation, but leads to defects in cell spreading and focal adhesion reinforcement. Furthermore, it could be shown that mechanical resilient linkages through talin-1 in focal adhesions are important for extracellular rigidity sensing.Taken together this work provides strong evidence that talin-1 mediated mechanical linkage between the extracellular matrix and the actin cytoskeleton is essential in mechanosignaling processes. For the first time it could be shown that talin-1 is subject to pN forces in living cells and that the force transmission is indeed dependent on f-actin and vinculin association with talin-1