Knowledge-based approaches for understanding structure-dynamics-function relationship in proteins

Proteins accomplish their functions through conformational changes, often brought about by changes in environmental conditions or ligand binding. Predicting the functional mechanisms of proteins is impossible without a deeper understanding of conformational transitions. Dynamics is the key link between the structure and function of proteins. The protein data bank (PDB) contains multiple structures of the same protein, which have been solved under different conditions, using different experimental methods or in complexes with different ligands. These alternate conformations of the same protein (or similar proteins) can provide important information about what conformational changes take place and how they are brought about. Though there have been multiple computational approaches developed to predict dynamics from structure information, little work has been done to exploit this apparent, but potentially informative, redundancy in the PDB. In this work I bridge this gap by exploring various knowledge-based approaches to understand the structure-dynamics relationship and how it translates into protein function. First, a novel method for constructing free energy landscapes for conformational changes in proteins is proposed by combining principal motions with knowledge-based potential energies and entropies from coarse-grained models of protein dynamics. Second, an innovative method for computing knowledge-based entropies for proteins using an inverse Boltzmann approach is introduced, similar to the manner in which statistical potentials were previously extracted. We hypothesize that amino acid contact changes observed in the course of conformational changes within a large set of proteins can provide information about local pairwise flexibilities or entropies. By combining this new entropy measure with knowledge-based potential functions, we formulate a knowledge-based free energy (KBF) function that we demonstrate outperforms other statistical potentials in its ability to identify native protein structures embedded with sets of decoys. Third, I apply the methods developed above in collaboration with experimentalists to understand the molecular mechanisms of conformational changes in several protein systems including cadherins and membrane transporters. This work introduces several ways that the huge data in the PDB can be utilized to understand the underlying principles behind the structure-dynamics-function relationships of proteins. Results from this work have several important applications in structural bioinformatics such as structure prediction, molecular docking, protein engineering and design. In particular, the new KBFs developed in this dissertation have immediate applications in emerging topics such as prediction of 3D structure from coevolving residues in sequence alignments as well as in identifying the phenotypic effects of mutants

Investigating the structure-dynamics-function relationship in antibodies

The paradigm that connects sequence, structure and function in proteins has been revisited in recent years, opening new perspectives on the importance of dynamics [1]. In this work we tackle this issue through the analysis of all-atom molecular dynamics (MD) simulations, with the final objective of correlating motions and structural features. We first characterize the dynamics of an antibody, through 2 μs of all-atom molecular dynamics simulations, to investigate the correlation between structural features and the flexibility of the molecule. Subsequently we perform 2 μs of all-atom MD simulations of the same antibody bound to its antigen, to investigate the changes in dynamics. We analyzed the simulations through various different techniques among which we highlight the power of those based on the calculation of the information transfer between different amino acids [3]. These types of measurements allow us to identify significant correlations among protein regions, providing clues on the mechanism of protein function. The investigations carried out in this work also serve as a guide in the identification of those structural patterns whose preservation is necessary in the construction of coarse-grained models. Overall this study is meant as a starting point for the application of a multi-scale method to biologically relevant macromolecules. [1] Hensen, U. et al. (2012). Exploring protein dynamics space: the dynasome as the missing link between protein structure and function. PloS one, 7(5).[2] Scapin, G. et al. (2015). Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab. Nat Struct Mol Biol, 22(12):953-8.[3] Bowerman, S., and J. Wereszczynski. (2016) Detecting Allosteric Networks Using Molecular Dynamics Simulation. Methods in enzymology. 578. 429-47

Exploring Structure-Dynamics-Function Relationship in Proteins, Protein: Ligand and Protein: Protein Systems through Computational Methods

The study focuses on understanding the dynamic nature of interactions between molecules and macromolecules. Molecular modeling and simulation technologies are employed to understand how the chemical constitution of the protein, specific interactions and dynamics of its structure provide the basis of its mechanism of function. The structure-dynamics-function relationship is investigated from quantum to macromolecular-assembly level, with applications in the field of rationale drug discovery and in improving efficiency of renewable sources of energy. Results presented include investigating the role of dynamics in the following: 1) In interactions between molecules: analyzing dynamic nature of a specific non-covalent interaction known as “anion-π [pi]” in RmlC protein. 2) In interactions between molecules and macromolecules: defining the structural basis of testosterone activation of GPRC6A. 3) In disrupting the function using specific substrate interactions: incorporating protein dynamics and flexibility in structure-based drug-discovery approach targeting the prothrombinase coagulation complex. 4) In interactions between macromolecules: elucidating the protein-protein binding and dynamics of electron-transport proteins, Ferrodoxin and Cytochrome c6, with Cyanobacterial Photosystem I

Dynamic control of selectivity in the ubiquitination pathway revealed by an ASP to GLU substitution in an intra-molecular salt-bridge network

Ubiquitination relies on a subtle balance between selectivity and promiscuity achieved through specific interactions between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Here, we report how a single aspartic to glutamic acid substitution acts as a dynamic switch to tip the selectivity balance of human E2s for interaction toward E3 RING-finger domains. By combining molecular dynamic simulations, experimental yeast-two-hybrid screen of E2-E3 (RING) interactions and mutagenesis, we reveal how the dynamics of an internal salt-bridge network at the rim of the E2-E3 interaction surface controls the balance between an “open”, binding competent, and a “closed”, binding incompetent state. The molecular dynamic simulations shed light on the fine mechanism of this molecular switch and allowed us to identify its components, namely an aspartate/glutamate pair, a lysine acting as the central switch and a remote aspartate. Perturbations of single residues in this network, both inside and outside the interaction surface, are sufficient to switch the global E2 interaction selectivity as demonstrated experimentally. Taken together, our results indicate a new mechanism to control E2-E3 interaction selectivity at an atomic level, highlighting how minimal changes in amino acid side-chain affecting the dynamics of intramolecular salt-bridges can be crucial for protein-protein interactions. These findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship and open new possibilities for control and fine-tuning of protein interaction selectivity

Functional Rotation of the Transporter AcrB: Insights into Drug Extrusion from Simulations

The tripartite complex AcrAB-TolC is the major efflux system in Escherichia coli. It extrudes a wide spectrum of noxious compounds out of the bacterium, including many antibiotics. Its active part, the homotrimeric transporter AcrB, is responsible for the selective binding of substrates and energy transduction. Based on available crystal structures and biochemical data, the transport of substrates by AcrB has been proposed to take place via a functional rotation, in which each monomer assumes a particular conformation. However, there is no molecular-level description of the conformational changes associated with the rotation and their connection to drug extrusion. To obtain insights thereon, we have performed extensive targeted molecular dynamics simulations mimicking the functional rotation of AcrB containing doxorubicin, one of the two substrates that were co-crystallized so far. The simulations, including almost half a million atoms, have been used to test several hypotheses concerning the structure-dynamics-function relationship of this transporter. Our results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. Furthermore, we provide strong evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. A concerted opening of the channel between the binding pocket and the gate further favors the displacement of the drug. This microscopically well-funded information allows one to identify the role of specific amino acids during the transitions and to shed light on the functioning of AcrB

Spectroscopic Studies of Peptide-Membrane Interactions

Understanding the structure-dynamics-function relationship is a fundamental motivation for studying how proteins fold. Over the past several decades, significant progress has been made in elucidating the folding energy landscapes and dynamics of soluble, globular proteins. In contrast, the folding kinetics and mechanisms of membrane proteins are much less studied and understood, due in part to the fact that they reside in the heterogeneous and complex membrane environment. To provide new mechanistic insights into membrane protein folding, herein we studied the folding kinetics of the influenza hemagglutinin fusion peptide (HAfp), which folds into a representative helix-turn-helix structure in model membranes. Our stopped-flow fluorescence and fluorescence resonance energy transfer (FRET) kinetics, obtained at different peptide-to-lipid ratios, support a parallel mechanism for membrane-peptide binding, wherein folding can occur either before or after membrane binding, but prior to membrane insertion. Thus, this result underscores the importance of the water-membrane interfacial region in mediating the process of folding, at least for short peptides. In turn, the association of the peptide to the interfacial region could induce local and global structural changes in the membrane. To help better characterize peptide-induced membrane structural changes as well as how cell penetrating peptides translocate across membranes, the second portion of this thesis was devoted to method development. Using two antimicrobial peptides and a cell penetrating peptide as examples, we showed that diffusion measurements via fluorescence correlation spectroscopy (FCS), can be used to `image\u27 peptide-induced lipid domain formation in model membranes and to elucidate the mechanism of peptide translocation

Computer-Based Screening of Functional Conformers of Proteins

A long-standing goal in biology is to establish the link between function, structure, and dynamics of proteins. Considering that protein function at the molecular level is understood by the ability of proteins to bind to other molecules, the limited structural data of proteins in association with other bio-molecules represents a major hurdle to understanding protein function at the structural level. Recent reports show that protein function can be linked to protein structure and dynamics through network centrality analysis, suggesting that the structures of proteins bound to natural ligands may be inferred computationally. In the present work, a new method is described to discriminate protein conformations relevant to the specific recognition of a ligand. The method relies on a scoring system that matches critical residues with central residues in different structures of a given protein. Central residues are the most traversed residues with the same frequency in networks derived from protein structures. We tested our method in a set of 24 different proteins and more than 260,000 structures of these in the absence of a ligand or bound to it. To illustrate the usefulness of our method in the study of the structure/dynamics/function relationship of proteins, we analyzed mutants of the yeast TATA-binding protein with impaired DNA binding. Our results indicate that critical residues for an interaction are preferentially found as central residues of protein structures in complex with a ligand. Thus, our scoring system effectively distinguishes protein conformations relevant to the function of interest

The Development Of Unnatural Amino Acid-Based Probes And Methods For Biological Studies

Proteins form a diverse ensemble of dynamic structures to carry out all life-sustaining functions. Therefore, many efforts have gone into studying the structure-dynamics-function relationship of proteins using a wide range of techniques, including fluorescence and infrared (IR) spectroscopies. While very useful, intrinsic fluorescence and IR signals arising from the natural amino acid side chains within the protein are often insufficient or unable to provide the information needed to understand the biological question of interest. To this end, various extrinsic spectroscopic probes, such as fluorescent dyes, have been used to increase the information content in specific measurements and applications. However, incorporation of a foreign moiety into any protein unavoidably affects its native structure and dynamics; hence effort must be made to reduce such perturbation. In this regard, the overarching aim of this thesis is to develop novel spectroscopic probes based on scaffolds of natural amino acids (NAAs). Because of their small size and similarity to NAAs, such unnatural amino acid-based (UAA-based) probes are expected to be minimally perturbing. Specifically, we show that (1) 4-cyanotryptophan (4CN-Trp) is a blue fluorescent amino acid useful for fluorescence microscopy applications; (2) 4CN-Trp and DiO (a common dye used to stain membranes) are a useful FRET pair to study peptide-membrane interactions; (3) 4CN-Trp, and tryptophan constitutes a dual FRET-PET pair which was used to study peptide end-to-end termini interactions and protein ligand-binding; and (4) the functional group of 4CN-Trp, 4-cyanoindole can be used in the form of a nucleoside as a dual fluorescence-IR reporter for DNA-protein studies. Furthermore, we extended applications of previously known UAAs and showed (5) p-cyanophenylalanine is useful as a fluorescence-based pH sensor which we used to determine peptide pKa’s and peptide membrane penetration kinetics and (6) we use a simple synthetic method for post-translationally installing an ester moiety on to proteins via cysteine alkylation as an UAA-based vibrational probe in proteins to study fibril formation and protein-ligand interaction

Characterization of the C-terminal binding domain from bacterial Enzyme I

Modulation of enzyme structure and flexibility by substrate/ligand binding provides an important source of enzyme function regulation. Unfortunately, our understanding of the fundamental mechanisms coupling protein dynamics to biological function is still largely incomplete, therefore limiting our ability to harness protein conformational dynamics in order to regulate enzymatic activity. Here we couple variable temperature (VT) NMR, particularly relaxation dispersion experiments, X-ray crystallography, computer simulations, protein engineering, and enzyme kinetic assays to explore the role of structural heterogeneity and conformational disorder in regulation of the C-terminal substrate binding domain (EIC) of bacterial Enzyme I (EI). In particular, we investigate the relationship between structure, conformational dynamics, and biological function of four EIC constructs: the wild type mesophilic enzyme (eEIC), a thermophilic homologue (tEIC), and two hybrid constructs engineered by incorporating the active site loops of the mesophilic enzyme into the scaffold of the thermophilic enzyme (etEIC), and vice versa (teEIC). Through this characterization we provide evidence that the four EIC constructs are structurally similar and that holo EIC undergoes an exchange between a disordered expanded inactive state and a more ordered compact active state. Furthermore, we report that the population of the active state dictates the effective turnover number and that this functional regulation is achieved by tuning the thermodynamic balance between the active and inactive states providing rational for thermal adaption (i.e. why thermophilic homologues exhibit lower activity than their mesophilic counterpart at low temperatures but increased activity comparable to its mesophilic homologue at higher temperatures). We demonstrate that altering thermal stability, conformational flexibility, and enzymatic activity through the hybridization of mesophilic/thermophilic enzyme pairs is a promising strategy for protein engineering in the field of biotechnology