Long-Range Intra-Protein Communication Can Be Transmitted by Correlated Side-Chain Fluctuations Alone

Allosteric regulation is a key component of cellular communication, but the way in which information is passed from one site to another within a folded protein is not often clear. While backbone motions have long been considered essential for long-range information conveyance, side-chain motions have rarely been considered. In this work, we demonstrate their potential utility using Monte Carlo sampling of side-chain torsional angles on a fixed backbone to quantify correlations amongst side-chain inter-rotameric motions. Results indicate that long-range correlations of side-chain fluctuations can arise independently from several different types of interactions: steric repulsions, implicit solvent interactions, or hydrogen bonding and salt-bridge interactions. These robust correlations persist across the entire protein (up to 60 Å in the case of calmodulin) and can propagate long-range changes in side-chain variability in response to single residue perturbations

Type II Kinase Inhibitors Show an Unexpected Inhibition Mode against Parkinson’s Disease-Linked LRRK2 Mutant G2019S

A number of well-known type II inhibitors (ATP-noncompetitive) that bind kinases in their DFG-out conformation were tested against wild-type LRRK2 and the most common Parkinson’s disease-linked mutation, G2019S. We found that traditional type II inhibitors exhibit surprising variability in their inhibition mechanism between the wild type (WT) and the G2019S mutant of LRRK2. The type II kinase inhibitors were found to work in an ATP-competitive fashion against the G2019S mutant, whereas they appear to follow the expected noncompetitive mechanism against WT. Because the G2019S mutation lies in the DXG motif (DYG in LRRK2 but DFG in most other kinases) of the activation loop, we explored the structural consequence of the mutation on loop dynamics using an enhanced sampling method called metadynamics. The simulations suggest that the G2019S mutation stabilizes the DYG-in state of LRRK2 through a series of hydrogen bonds, leading to an increase in the conformational barrier between the active and inactive forms of the enzyme and a relative stabilization of the active form. The conformational bias toward the active form of LRRK2 mutants has two primary consequences. (1) The mutant enzyme becomes hyperactive, a known contributor to the Parkinsonian phenotype, as a consequence of being “locked” into the activated state, and (2) the mutation creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP-competitive fashion. Our results suggest that developing type II inhibitors, which are generally considered superior to type I inhibitors because of desirable selectivity profiles, might be especially challenging for the G2019S LRRK2 mutant

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

Molecular Dynamics Investigations of Structural Conversions in Transformer Proteins

Multifunctional proteins that undergo major structural changes to perform different functions are known as “Transformer Proteins”, which is a recently identified class of proteins. One such protein that shows a remarkable structural plasticity and has two distinct functions is the transcription antiterminator, RfaH. Depending on the interactions between its N-terminal domain and its C-terminal domain, the RfaH CTD exists as either an all-α-helix bundle or all-β-barrel structure. Another example of a transformer protein is the Ebola virus protein VP40 (eVP40), which exists in different conformations and oligomeric states (dimer, hexamer, and octamer), depending on the required function.I performed Molecular Dynamics (MD) computations to investigate the structural conversion of RfaH-CTD from its all-a to all-b form. I used various structural and statistical mechanics tools to identify important residues involved in controlling the conformational changes. In the full-length RfaH, the interdomain interactions were found to present the major barrier in the structural conversion of RfaH-CTD from all-a to all-b form. I mapped the energy landscape for the conformational changes by calculating the potential of mean force using the Adaptive Biasing Force and Jarzynski Equality methods. Similarly, the interdomain salt-bridges in the eVP40 protomer were found to play a critical role in domain association and plasma membrane (PM) assembly. This molecular dynamic simulation study is supported by virus like particle budding assays investigated by using live cell imaging that highlighted the important role of these saltbridges. I also investigated the plasma membrane association of the eVP40 dimer in various PM compositions and found that the eVP40 dimer readily associates with the PM containing POPS and PIP2 lipids. Also, the CTD helices were observed to be important in stabilizing the dimer-membrane complex. Coarse-grained MD simulations of the eVP40 hexamer and PM system revealed that the hexamer enhances the PIP2 lipid clustering at the lower leaflet of the PM. These results provide insight on the critical steps in the Ebola virus life cycle

A conformational analysis of signal peptides

A thesis submitted to the Faculty of Science University of the Witwatersrand in fulfillment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 1998.Conformational analysis of portions of functionally-active and functionally-inactive signal peptides (incorporating the wild-type and mutants thereof) has been performed using a variety of computational prediction techniques based on both statistics and molecular mechanics. Molecular mechanics conformational studies are generally plagued by the problem of combinatorial explosion; this problem was addressed with a systematic searching procedure as well as a recently developed genetic algorithm, both utilising tile ECEPP/3 force field. The genetic algorithm, in combination with a gradient minimiser, proved to be successful in finding low-energy conformations for each peptide sequence studied. Analysis was performed in both simulated hydrophobic and hydrophilic environments, under distance-constraints. The molecular mechanics results and statistical predictions generated from the study were compared With existing experimental observations. The reliability of statistical predictions proved to be dependent on prediction method; the more consistent predictions were produced by methods based on membrane proteins, as opposed to those based on globular proteins. The physical property of hydrophobicity of signal peptide sequences, explored in these statistical predictions, was determined to be an important factor in relating sequence to functional activity. Molecular mechanics calculations produced either interrupted or non interrupted a-helical secondary structures both for functionally-efficient and for functionally-inefficient signal peptides, indicating that cc-helixformation alone cannot be correlated with protein export competence. It was concluded from our overall results that both a-helicity and hydrophobicity are required for the efficient functioning of signal peptides.AC201

Molecular dynamics simulations in photosynthesis

Photosynthesis is regulated by a dynamic interplay between proteins, enzymes, pigments, lipids, and cofactors that takes place on a large spatio-temporal scale. Molecular dynamics (MD) simulations provide a powerful toolkit to investigate dynamical processes in (bio)molecular ensembles from the (sub)picosecond to the (sub)millisecond regime and from the Å to hundreds of nm length scale. Therefore, MD is well suited to address a variety of questions arising in the field of photosynthesis research. In this review, we provide an introduction to the basic concepts of MD simulations, at atomistic and coarse-grained level of resolution. Furthermore, we discuss applications of MD simulations to model photosynthetic systems of different sizes and complexity and their connection to experimental observables. Finally, we provide a brief glance on which methods provide opportunities to capture phenomena beyond the applicability of classical MD

Multiplexed single molecule observation and manipulation of engineered biomolecules

Molecular processes in organisms are often enabled by structural elements resilient to mechanical forces. For instance, the microbial and hierarchical cellulosome protein system comprises enzymes and the receptor-ligand complexes Cohesin-Dockerin (Coh-Doc), that act in concert for the efficient hydrolysis of plant polysaccharides. The Coh-Doc complexes can withstand remarkably high forces to keep host cells and enzymes bound to their substrates in the extreme environmental conditions the microorganisms frequently live in. This work focuses on the investigation of mechanical stability of such biomolecules on the single-molecule level. The highly symmetric binding interface of the Coh-Doc type I complex from Clostridium thermocellum, enables two different binding conformations withcomparable affinity and similar strength. I was able to show that both conformations exist in the wild-type molecules and are occupied under native conditions. I further characterized one of the strongest non-covalent protein complexes known, Coh-Doc type III from Ruminococcus flavefaciens by elucidating the pivotal role of the adjacent xModule domain for the mechanical stabilization of the whole complex and the role of the bimodal rupture force distribution. Such large forces impair accuracy of measured contour length increments in unfolding studies by inducing conformational changes in poly-ethylene glycol (PEG) linkers in aqueous buffer systems. This problemwas solved by introducing elastin-like polypeptides (ELP) as surface tethers. Having a peptide backbone similar to that of unfolded proteins, ELP linkers do not alter accuracy of the single-molecule force spectroscopy (SMFS) assay. To provide high throughput and precise comparability, I worked on a microfluidic platform for the in vitro protein synthesis and immobilization. The Coh-Doc system was hereby integrated as a binding handle for multiplexed measurements of mechanostability. Employing a single AFM probe to measure multiple different molecules facilitates force precision required to shed light onto molecular mechanisms down to the level of single amino acids. I also applied the Coh-Doc complex to a purely protein based single-molecule cut and paste assay for the bottom-up assembly of molecular systems for quick phenotyping of spatial arrangements. With this system, interactions in enzymatic synergies can be studied by defined positioning patterns on the single molecule level. To understand and design force responses of complex systems, I complemented the investigation of protein systems with SMFS studies on DNA Origami structures. The results of SMFS on DNA were compared to a simulation framework. Despite their difference in force loading rates, both methods agree well within their results, enabling better fundamental understanding of complex molecular superstructures.Molekulare Prozesse in Organismenwerden oft von Strukturelementen ermöglicht, die mechanischen Kräften standhalten können. Ein Beispiel hierfür ist das mikrobielle und hierarchisch aufgebaute Proteinsystem des Zellulosoms. Enzyme und die Rezeptor-Liganden Komplexe Cohesin-Dockerin (Coh-Doc) arbeiten hierbei für die effiziente Hydrolyse von pflanzlichen Polysacchariden zusammen. Die Coh-Doc Komplexe können bemerkenswerten Kräften standhalten, um in den extremen Umweltbedingungen, in denen die Mikroorganismen teilweise leben, die Wirtszellen und Enzyme an ihre Substrate binden zu können. Die vorliegende Arbeit untersucht den Einfluss von mechanischer Kraft auf solche Biomoleküle mittels Einzelmolekülmessungen. Die hohe Symmetrie des Bindeinterfaces des Coh-Doc Typ I Komplexes aus Clostridium thermocellum ermöglicht zwei verschiedene Konformationen, die vergleichbare Affinität und Stärke aufweisen. Im Rahmen dieser Arbeit konnte ich beide in denWildtyp-Molekülen und unter nativen Bedingungen nachweisen. Eines der stärksten bekannten nicht-kovalenten Rezeptor-Liganden Systeme, Coh- Doc Typ III aus Ruminococcus flavefaciens wurde charakterisiert, und die Kernrolle des benachbarten xModuls für die Stabilität des gesamten Komplexes sowie die Rolle der bimodalen Kraftverteilung untersucht. Solch hohe Kräfte vermindern die Genauigkeit der gemessenenKonturlängeninkremente von Proteinentfaltungen, indem sie Konformationsänderungen der Poly- Ethylenglykol (PEG) Oberflächenanker in wässrigen Puffersystemen verursachen. Mit Elastin-ähnlichen Polypeptiden (ELP) als Anker wurde dieses Problem gelöst: durch die Ähnlichkeit des Peptid-Rückgrates von ELPs mit dem entfaltener Proteine beeinflussen diese die Genauigkeit des Experiments nicht. Für die Optimierung von Messdurchsatz und Vergleichbarkeit entwickelte ich an einer Mikrofluidik-Plattform zur in vitro Proteinsynthese und -immobilisierung. Das Coh-Doc System wurde hierbei als Binde-Molekül für gemultiplexte Messungen integriert. Die dadurch ermöglichte Nutzung einer einzigen AFM Messsonde für die Messung verschiedener Moleküle erlaubt die nötige Kraftpräzision, um molekulare Mechanismen bis auf die Ebene einzelner Aminosäuren aufzuklären. Des weiteren habe ich den Coh-Doc Komplex in einem rein auf Proteininteraktionen basierten ’Cut and Paste’ Assay für den modularen Aufbau molekularer Systeme implementiert. Dieses ermöglicht schnelle Phänotypisierung geometrischer Anordnunungen und die Untersuchung von Wechselwirkung zwischen Enzymen mittels definierter Positionierung auf Einzelmolekülebene. Um die Kraftantwort komplexer Systeme besser verstehen und letztendlich gestalten zu können, ergänzte ich die Untersuchung von Proteinsystemen mit derer von DNA-Origami Strukturen. Die Ergebnisse der Kraftspektroskopie an DNA wurden mit Computersimulationen verglichen, und trotz des großen Unterschieds ihrer Ladungsraten stimmen beide Methoden gut überein. Dadruch legen sie die Grundlagen für ein besseres Verständnis komplexer molekularer Superstrukturen

Modelling the binding mode of macrocycles: Docking and conformational sampling

Drug discovery is increasingly tackling challenging protein binding sites regarding molecular recognition and druggability, including shallow and solvent-exposed protein-protein interaction interfaces. Macrocycles are emerging as promising chemotypes to modulate such sites. Despite their chemical complexity, macrocycles comprise important drugs and offer advantages compared to non-cyclic analogs, hence the recent impetus in the medicinal chemistry of macrocycles. Elaboration of macrocycles, or constituent fragments, can strongly benefit from knowledge of their binding mode to a target. When such information from X-ray crystallography is elusive, computational docking can provide working models. However, few studies have explored docking protocols for macrocycles, since conventional docking methods struggle with the conformational complexity of macrocycles, and also potentially with the shallower topology of their binding sites. Indeed, macrocycle binding mode prediction with the mainstream docking software GOLD has hardly been explored. Here, we present an in-depth study of macrocycle docking with GOLD and the ChemPLP scores. First, we summarize the thorough curation of a test set of 41 protein-macrocycle X-ray structures, raising the issue of lattice contacts with such systems. Rigid docking of the known bioactive conformers was successful (three top ranked poses) for 92.7% of the systems, in absence of crystallographic waters. Thus, without conformational search issues, scoring performed well. However, docking success dropped to 29.3% with the GOLD built-in conformational search. Yet, the success rate doubled to 58.5% when GOLD was supplied with extensive conformer ensembles docked rigidly. The reasons for failure, sampling or scoring, were analyzed, exemplified with particular cases. Overall, binding mode prediction of macrocycles remains challenging, but can be much improved with tailored protocols. The analysis of the interplay between conformational sampling and docking will be relevant to the prospective modelling of macrocycles in general

Computational Perspective on Intricacies of Interactions, Enzyme Dynamics and Solvent Effects in the Catalytic Action of Cyclophilin A

Cyclophilin A (CypA) is the well-studied member of a group of ubiquitous and evolutionarily conserved families of enzymes called peptidyl–prolyl isomerases (PPIases). These enzymes catalyze the cis-trans isomerization of peptidyl-prolyl bond in many proteins. The distinctive functional path triggered by each isomeric state of peptidyl-prolyl bond renders PPIase-catalyzed isomerization a molecular switching mechanism to be used on physiological demand. PPIase activity has been implicated in protein folding, signal transduction, and ion channel gating as well as pathological condition such as cancer, Alzheimer’s, and microbial infections. The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerization by CypA has been the target of intense research. Normal and accelerated molecular dynamic simulations were carried out to understand the catalytic mechanism of CypA in atomistic details. The results reaffirm transition state stabilization as the main factor in the astonishing enhancement in isomerization rate by enzyme. The ensuing intramolecular polarization, as a result of the loss of pseudo double bond character of the peptide bond at the transition state, was shown to contribute only about −1.0 kcal/mol to stabilizing the transition state. This relatively small contribution demonstrates that routinely used fixed charge classical force fields can reasonably describe these types of biological systems. The computational studies also revealed that the undemanding exchange of the free substrate between β- and α-helical regions is lost in the active site of the enzyme, where it is mainly in the β-region. The resultant relative change in conformational entropy favorably contributes to the free energy of stabilizing the transition state by CypA. The isomerization kinetics is strongly coupled to the enzyme motions while the chemical step and enzyme–substrate dynamics are in turn buckled to solvent fluctuations. The chemical step in the active site of the enzyme is therefore not separated from the fluctuations in the solvent. Of special interest is the nature of catalysis in a more realistic crowded environment, for example, the cell. Enzyme motions in such complicated medium are subjected to different viscosities and hydrodynamic properties, which could have implications for allosteric regulation and function