Molecular Dynamics Simulations

figurasGroEL is an ATP dependent molecular chaperone that promotes the folding of a large number of substrate proteins in E. coli. Large-scale conformational transitions occurring during the reaction cycle have been characterized from extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in the allosteric response to ATP and the nucleotide-driven reaction cycle are not completely established. Here we describe extensive (in total long) unbiased molecular dynamics (MD) simulations that probe the response of GroEL subunits to ATP binding. We observe nucleotide dependent conformational transitions, and show with multiple 100 ns long simulations that the ligand-induced shift in the conformational populations are intrinsically coded in the structure-dynamics relationship of the protein subunit. Thus, these simulations reveal a stabilization of the equatorial domain upon nucleotide binding and a concomitant “opening” of the subunit, which reaches a conformation close to that observed in the crystal structure of the subunits within the ADP-bound oligomer. Moreover, we identify changes in a set of unique intrasubunit interactions potentially important for the conformational transition.The Norwegian Research Council is acknowledged for CPU resources granted through the NOTUR supercomputing program (http://www.notur.no/) and Bergen Center for Computational Science for providing powerful computer facilities (http://www.bccs.uni.no/). Work at CSIC/UPV/EHU was financed by MICINN (Grant BUF2007-64452). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Molecular simulations of chaperonins

Chaperonins are a class of cage-like molecular machines that assist the folding of polypeptides by binding and releasing non-native substrates into their inner cavity, where sequestered from the medium the substrate can fold to its native state. The main goal of this work is to understand the structure-function relationship of chaperonin. Here we study two major aspects of this relationship: (1) thermodynamics of protein folding inside the chaperonin cavity and (2) conformational changes of the E. coli chaperonin GroEL in its reaction cycle. We have studied the thermodynamics of protein folding, confined in the chaperonin cavity using the simple HP model of protein undergoing the coil-to-globule transition. Using the Wang-Landau method we have quantified the increase in thermal stability of a native state in a spherical confining geometry, measured as an increase in melting temperature with decreasing radius of confining sphere. We are the first to show that the t -> r transition in GroEL subunit occurs spontaneously using unbiased molecular dynamics simulation. The transition pathway is shown to lie along low frequency quasi-harmonic modes of vibration. We are also the first to observe the spontaneous insertion of Ala480 into the empty nucleotide binding pocket, required for negative interring cooperativity.Ph.D., Chemical Engineering -- Drexel University, 200

Coherent Conformational Degrees of Freedom as a Structural Basis for Allosteric Communication

Conformational changes in allosteric regulation can to a large extent be described as motion along one or a few coherent degrees of freedom. The states involved are inherent to the protein, in the sense that they are visited by the protein also in the absence of effector ligands. Previously, we developed the measure binding leverage to find sites where ligand binding can shift the conformational equilibrium of a protein. Binding leverage is calculated for a set of motion vectors representing independent conformational degrees of freedom. In this paper, to analyze allosteric communication between binding sites, we introduce the concept of leverage coupling, based on the assumption that only pairs of sites that couple to the same conformational degrees of freedom can be allosterically connected. We demonstrate how leverage coupling can be used to analyze allosteric communication in a range of enzymes (regulated by both ligand binding and post-translational modifications) and huge molecular machines such as chaperones. Leverage coupling can be calculated for any protein structure to analyze both biological and latent catalytic and regulatory sites

Analiza porównawcza własności fizycznych miejsc wiązania antybiotyków aminoglikozydowych w RNA i białkach

Aminoglycoside antibiotics have been in use for more than 60 years, helping combat severe bacterial infections. Due to this long time of usage, more and more bacteria become resistant to one or several drugs from this group. This spread of resistant species is alarming and additionally, there is little knowledge about the mechanisms of bacterial resistance. In order to broaden our understanding of how bacteria combat aminoglycosides, we performed computer simulations of various molecules that bind aminoglycosides in a bacterial cell: (i) the primary binding site, called the A-site and located in ribosomal RNA, wild type and with mutations that decrease the aminoglycoside binding affinity; and (ii) the aminoglycoside modifying enzymes (AMEs), which are produced by bacteria to inactivate these drugs. The mutations of the RNA A-site were chosen based on previous experimental studies on whole bacteria. These studies showed that even single base substitutions were sufficient to make bacteria resistant, but did not explain how this resistance was gained on an atomic level. There are many AMEs and they vary a lot among themselves, yet they all have a narrow specificity towards aminoglycosides, which are quite homogeneous group. The two main questions we have posed in our research are: (i) what are the physical grounds of bacteria becoming less susceptible to aminoglycosides due to RNA A-site mutations; and (ii) how different AMEs attract aminoglycosides and interact with them? We performed all-atom molecular dynamics (MD) simulations of the A-site model with selected mutations and of AME representatives. In addition, the complexes of these biomolecules with aminoglycosides were simulated. For comparison, we also performed simulations of the wild type A-site model and of the aminoglycosides in water. We used various biophysical methods to analyze these simulations and to study: internal dynamics of the biomolecules; electrostatic potential, shape, and volume of the binding pockets; types of interactions with aminoglycosides; and changes in conformations of aminoglycosides. In addition, we developed and implemented an algorithm that helps describe molecular motions. We found that different A-site mutations affect different features of the RNA binding site. Some of them changed the mobility of the nucleic bases, and therefore the shape of the A-site was altered. Other mutations changed the electrostatic potential inside the binding site, thus making it almost unrecognizable to aminoglycosides. The study of AMEs showed that apart from their structural and sequence-related diversity, they differ in the internal movement patterns. However, these enzymes interact with aminoglycosides very similarly, using mainly electrostatic interactions. Interestingly, we noticed that these interactions were copied from the RNA:aminoglycoside complex. Our findings were in agreement with experimental studies and also helped to explain some of their outcomes. The results presented in this dissertation may help design new antibiotics that would overcome the bacterial resistance.Od ponad 60 lat antybiotyki aminoglikozydowe są z powodzeniem stosowane w szpitalach przeciwko ciężkim infekcjom bakteryjnym. Jednak pojawianie się coraz większej liczby przypadków bakterii opornych na stosowane aminoglikozydy sprawia, że badania mechanizmów oporności u bakterii stają kluczowe w dalszej skutecznej walce z infekcjami tego typu. Przeprowadziłam komputerowe symulacje biomolekuł, które oddziałują z antybiotykami aminoglikozydowymi we wnętrzu komórek bakteryjnych. Badanymi obiektami są: (i) główne miejsce wiązania aminoglikozydów, zwane miejscem A, w rybosomalnym RNA; natywne oraz z mutacjami powodującymi wzrost oporności u bakterii; a także (ii) enzymy modyfikujące aminoglikozydy (ang. aminoglycoside modifying enzymes, AME}), produkowane przez bakterie w celu chemicznej dezaktywacji tych leków. Motywacją do badań nad zmutowanym miejscem A był brak informacji o zmianach jakie zachodzą w fizycznych własnościach miejsca A po różnych zamianach nukleotydów. Wiadomo jakie mutacje prowadzą do oporności oraz że nawet pojedyncze zamiany nukleotydu mogą mieć bardzo wymierne skutki, ale nie wyjaśniono jakie są tego podstawy. Natomiast, w przypadku AME, celem prowadzenia symulacji było wyjaśnienie w jaki sposób ta grupa białek jest w stanie być jednocześnie bardzo zróżnicowana i wysoce specyficzna względem aminoglikozydów. Przeprowadziłam symulacje dynamiki molekularnej (MD) modelu miejsca A z wybranymi mutacjami oraz reprezentatywnych enzymów z trzech największych rodzin AME. Aby uzyskać opis oddziaływań między tymi miejscami wiążącymi a aminoglikozydami, przeprowadziłam również symulacje MD tych biomolekuł w kompleksach z wybranymi antybiotykami. W celu analizy symulacji użyłam metodologii z zakresu biofizyki teoretycznej. Badałam wiele własności fizykochemicznych wybranych biomolekuł i ich kompleksów, m.in.: dynamikę wewnętrzną, własności elektrostatyczne, kształt i objętość miejsc wiązania aminoglikozydów, a także rodzaje oddziaływań z aminoglikozydami. Ponadto, stworzyłam nową metodę analizy zmian konformacyjnych w molekułach, która dokonuje podziału biomolekuł na tzw. dynamiczne domeny, na podstawie danych pochodzących z symulacji lub eksperymentów. Z analizy symulacji rybosomalnego miejsca A wynika, że mutacje różnych zasad wpływają na różne własności fizyczne tego fragmentu RNA. W zależności od położenia mutowanej zasady, zmieniał się rozkład ładunków cząstkowych w miejscu wiążącym lub kształt tego miejsca. Mutacje wpływały również na dynamikę ruchów wewnętrznych miejsca A. Analiza symulacji cząsteczek AME wskazała, że oprócz różnorodności struktur trzeciorzędowych i sekwencji, występuje w tej grupie również różnorodność w ruchach wewnętrznych. Pomimo tych różnic, wszystkie enzymy oddziaływały z aminoglikozydami w bardzo podobny sposób, głównie elektrostatycznie. Ponadto, te oddziaływania wydają się być kopiowane z kompleksów, jakie aminoglikozydy tworzą z miejscem A. Rezultaty moich badań są zgodne z poprzednimi doniesieniami eksperymentalnymi, a także pomagają wyjaśnić niektóre z nich. Wyniki opisane w tej pracy mogą być podstawą do zaprojektowania zmodyfikowanych aminoglikozydów, które mogłyby być aktywne nawet wobec opornych bakterii

Complementary Mass Spectrometry Methods for Characterizing Protein Folding, Structure, and Dynamics

Proteins are involved in virtually every biochemical process. A comprehensive characterization of factors that govern protein function is essential for understanding the biomedical aspects of human health. This dissertation aims to develop complementary mass spectrometry-based methods and apply them to solve problems pertaining to the area of protein structure, folding and dynamics. ‎Chapter 1 uses fast photochemical oxidation of proteins (FPOP) to characterize partially disordered conformers populated under semi-denaturing conditions. In FPOP, ·OH generated by laser photolysis of H2O2 introduces oxidative modifications at solvent accessible side chains. By contrast, buried sites are protected from radical attack. Using apomyoglobin (aMb), it was demonstrated that under optimized conditions undesired can be almost completely eliminated and detailed structural information can be obtained. ‎Chapter 3 combines FPOP with submillisecond mixing to enable studying early events in protein folding. aMb served as a model system for these measurements. Spatially-resolved changes in solvent accessibility follow the folding process. Data revealed that early aMb folding events are driven by both local and sequence-remote docking of hydrophobic side chains. Assembly of a partially formed scaffold after 0.2 ms is followed by stepwise consolidation that ultimately yields the native state. The submillisecond mixer used improved the time resolution by a factor of 50 compared to earlier FPOP experiments. Submillisecond mixing in conjunction with slower mixing techniques help monitor completes folding pathways, from fractions of a millisecond all the way to minutes. ‎Chapter 4 uses ion mobility mass spectrometry (IM-MS) to explore the structural relationship between semi folded solution and gas phase protein conformers. Collision cross sections (CCSs) provide a measure of analyte size. Mb was used as model system because it follows a sequential unfolding pathway that comprises two partially disordered states. IM-MS data showed that the degree of gas phase unfolding is not strongly correlated with the corresponding solution. Gas phase unfolding as well as collapse events can lead to disparities between gaseous and solution structures for partially unfolded proteins. IM-MS data on non-native conformers should therefore be interpreted with caution. ‎Chapter 5 uses HDX-MS to examine the role of conformational dynamics for the function of multi-protein molecular machines such as FoF1 ATP synthase. HDX-MS monitors backbone deuteration kinetics in the presence of D2O. Disordered segments exchange more rapidly than those in tightly folded regions. Measurements of spatially-resolved deuterium are performed using LC-MS. It was found that the H-bonding network of key power transmission elements is insensitive to PMF-induced mechanical stress. Unexpectedly, HDX-MS reveals a pronounced destabilization of the g C-terminus during rotational catalysis under PMF. The behavior of g is attributed to kinetic friction within the apical rotor bearing

In-silico Investigation of Ion-Pumping Rotary A- and V-type ATPases: Structural and Dynamical Aspects

Advances in Molecular Biosciences have revolutionised the way we perceive and pursue current biological research. Dynamic, complex biomacromolecules constitute the essential components of Cells. Particularly proteins have been characterised as the workhorse molecules of life. Either as single chains or complexes of associated units, proteins participate in every biological process with a specific structural and/or functional role. Ion-pumping rotary ATPases is a large family of important membrane-bound protein nanomachines. In the current work we investigate structural and dynamical aspects of the A- and V-type rotary ATPases, related to functional dynamics, and propose a multiscale computational framework for their in-silico biophysical characterisation and the interpretation of low-resolution experimental data from electron microscopy in Chapter 3. For the first time we present results from explicit-solvent atomistic molecular dynamics simulations of the prokaryotic A-type peripheral stator stalk and central rotor axle, both being critical subunits involved in the mechanical coupling of the rotary ATPases in Chapter 4. Our simulation data reveal the presence of flexibility heterogeneity and demonstrate the dynamic nature of the peripheral stator stalk as a source of intact ATPase particle conformational variability. In Chapter 5 we show the presence of structural plasticity in the eukaryotic peripheral stator stalk of the V-ATPase and discuss possible implications for V-ATPase regulation. Overall, the wealth of information accessed with molecular-dynamics simulations allows the exploitation of atomistic information within the multiscale framework of Chapter 3 to be applied for the mechanical characterisation of rotary ATPases in future studies. In particular, atomistic data could serve as high-resolution information for future parameterisation of simplified coarse-grain models for all ATPase subunits and the construction of molecular models for the intact ATPases. We anticipate that our approach will contribute to elucidating the molecular origin of rotary ATPases’ conformational flexibility and its implications for the holoenzyme’s function and kinetic efficiency

A dynamics based analysis of allosteric modulation in heat shock proteins

The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies

A Bioinformatics Study of Protein Conformational Flexibility and Misfolding: a Sequence, Structure and Dynamics Approach

This PhD Thesis titled "A Bioinformatics Study of Protein Conformational Flexibility and Misfolding: a Sequence, Structure and Dynamics Approach" comprises the results and conclusions obtained by us from the study of three different but somehow related research projects, covering aspects of the phenomenon of protein local conformational instability, its relationship with protein function, evolvability and aggregation, and the effect of genetic variations on protein conformational instability related to Conformational Diseases. These projects include the prediction of putative prion proteins in complete proteomes and the study of prion biology from a genomic perspective, the prediction of conformationally unstable protein regions and the existence of a structural framework for linking conformational instability to folding and function, and the establishment of a rationale for assessing the connection among mutations and disease phenotypes in Conformational Diseases.Esta tesis doctoral comprende los resultados y conclusiones obtenidos por nosotros a partir del estudio de tres proyectos de investigación diferentes pero de alguna manera relacionados, cubriendo los aspectos del fenómeno de la inestabilidad conformacional local de la proteína, su relación con la función de la proteína, la capacidad de evolución y agregación, y el efecto de las variaciones genéticas en la inestabilidad conformacional de la proteína relacionados con las enfermedades conformacionales. Estos proyectos incluyen la predicción de presuntas proteínas priónicas en proteomas complejos y el estudio de la biología de priones desde una perspectiva genómica, la predicción de las regiones de proteínas conformacionalmente inestables y la existencia de un marco estructural para la vinculación de la inestabilidad conformacional del plegado y la función, y el establecimiento de una razón fundamental para la evaluación de la relación entre las mutaciones y fenotipos de la enfermedad en enfermedades conformacionales