Prediction of Protein–Protein Interactions Between Alsin DH/PH and Rac1 and Resulting Protein Dynamics

Alsin is a protein of 1,657 amino acids known for its crucial role in vesicular trafficking in neurons thanks to its ability to interact with two guanosine triphosphatases, Rac1 and Rab5. Evidence suggests that Rac1 can bind Alsin central region, composed by a Dbl Homology (DH) domain followed by a Pleckstrin Homology (PH) domain, leading to Alsin relocalization. However, Alsin three-dimensional structure and its relationship with known biological functions of this protein are still unknown. In this work, a homology model of the Alsin DH/PH domain was developed and studied through molecular dynamics both in the presence and in the absence of its binding partner, Rac1. Due to different conformations of DH domain, the presence of Rac1 seems to stabilize an open state of the protein, while the absence of its binding partner results in closed conformations. Furthermore, Rac1 interaction was able to reduce the fluctuations in the second conserved region of DH motif, which may be involved in the formation of a homodimer. Moreover, the dynamics of DH/PH was described through a Markov State Model to study the pathways linking the open and closed states. In conclusion, this work provided an all-atom model for the DH/PH domain of Alsin protein; moreover, molecular dynamics investigations suggested underlying molecular mechanisms in the signal transduction between Rac1 and Alsin, providing the basis for a deeper understanding of the whole structure–function relationship for Alsin protein

Simulation Of Auto-Inhibition Effect In Mev Ntail With Its Binding Partner XD

Master'sMASTER OF SCIENC

Solution structure of hMBD1 CXXC1

Methylation of CpG dinucleotides is the major epigenetic modification of mammalian DNA which results in the remodelling of transcriptionally active euchromatin to transcriptionally inactive heterochromatin. Recognition of methylated CpG by methylated DNA binding proteins, the MBD family, the Kaiso zinc finger family and the SRA domain proteins results in deacetylation and methylation of histone side chains through the recruitment of HDAC and HMT enzymes. Methylation of DNA is a heritable process ensuring Methylation dependant transcriptional repression is passed from mother to daughter cell during replication. Some of the proteins involved in this chromatin remodelling, MBD1, DNMT1, MLL, and CFP1 contain CXXC domains. hMBD1 contains 2 or 3 CXXC domains depending on the splice variant, with only the third CXXC domain shown to bind CpG dinucleotides. This thesis describes the work done to elucidate the structure of hMBD1 CXXC1 and to investigate hMBD1 CXXC12 di-domain by NMR spectroscopy and biochemical characterisation. The hMBD1 CXXC1 & CXXC12 domains were successfully over expressed in E. coli and purified. Unlabelled and uniformly 15N labelled proteins were produced for nuclear magnetic resonance (NMR) studies. Assignment of NMR spectra was carried out and constraints generated enabling structure determination of hMBD1 CXXC1 and to investigate the relationship between CXXC1 and CXXC2 of hMBD1. The solution structure of hMBD1 CXXC1 determined here was compared to the previously determined solution structure of hMLL CXXC in order to investigate their differences in DNA binding. NOE data from hMBD1 CXXC1 and CXXC12 are compared in order to investigate the domain structure of CXXC12. DNA titration of hMBD1 CXXC1 showed no significant interaction with a single CpG oligo while the loop region of hMBD1 CXXC1 differs significant in both structure and surface charge suggesting the loop region to be important for DNA binding. The recorded NOE data of hMBD CXXC12 suggests the two CXXC domains form a globular rather than a linear structur

Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches

Unlike other members of the methyl-cytosine binding domain (MBD) family, MBD4 serves as a potent DNA glycosylase in DNA mismatch repair specifically targeting mCpG/TpG mismatches arising from spontaneous deamination of methyl-cytosine. The protein contains an N-terminal MBD (MBD4MBD) and a C-terminal glycosylase domain (MBD4GD) separated by a long linker. This arrangement suggests that the MBD4MBD either directly augments enzymatic catalysis by the MBD4GD or targets the protein to regions enriched for mCpG/TpG mismatches. Here we present structural and dynamic studies of MBD4MBD bound to dsDNA. We show that MBD4MBD binds with a modest preference formCpG as compared to mismatch, unmethylated and hydroxymethylated DNA. We find that while MBD4MBD exhibits slow exchange between molecules of DNA (intermolecular exchange), the domain exhibits fast exchange between two sites in the same molecule of dsDNA (intramolecular exchange). Introducing a single-strand defect between binding sites does not greatly reduce the intramolecular exchange rate, consistent with a local hopping mechanism for moving along the DNA. These results support a model in which the MBD4MBD4 targets the intact protein to mCpG islands and promotes scanning by rapidly exchanging between successive mCpG sites which facilitates repair of nearby mCpG/TpG mismatches by the glycosylase domain

Structural studies of the DNA partitioning protein IncC from the plasmid RK2

Plasmid DNA partitioning is a crucial process for the transfer of at least a single copy of plasmid to the daughter cells during bacterial cell division. Partitioning for various low-copy number plasmids involves a DNA-binding protein (ParB), a centromere-like DNA site ( parS) and a ParA-family protein. Interestingly, the RK2 plasmid encodes two ParA proteins of different lengths. The longer protein is IncC1 (364 a.a), while IncC2 lacks a N- terminal domain of 105 amino acids (IncC NTD). The secondary structure of IncC NTD by NMR spectroscopy and other biophysical methods has been determined as random coil. It appears to bind DNA weakly and non-specifically. The expression and purification of IncC1 and IncC2 proteins was optimized. The two proteins and IncC NTD were characterized using various biophysical methods including Circular Dichroism, Analytical Ultracentrifugation, Small Angle X-ray Scattering, Size Exclusion Chromatography-Multi Angle Light Scattering, and EMSAs. Bacterial two hybrid assays and chemical crosslinking showed the two IncC proteins form homo- and hetero-dimers and interact with KorB protein. IncC1 and IncC2 proteins bind to DNA, non-specifically. IncC1 binds DNA weakly in the absence of nucleotides but IncC2 protein was found to bind DNA only in the presence of nucleotides (ADP, ATP)

Ligand recognition by the major urinary protein

Molecular Dynamics (MD) and Quartz Crystal Microbalance (QCM) techniques can provide unique insights into what drives protein-ligand association. The major urinary protein (MUP) binds small ligands in a deeply buried hydrophobic pocket. Detailed calorimetric studies have shown that ligand binding is driven by enthalpic effects, not entropic effects [1]. Previous studies have shown that this is due to 'dewetting' of the binding site cavity even in the absence of ligands, and have also characterised the complex changes in molecular flexibility that accompany ligand binding-features that may be correlated with NMR data [2]. Recent MD revealed the hydration effects of apo-MUP and also shown where certain regions of MUP become more flexible upon ligand binding. They have also shown a water molecule remains close to the tyrosine in the binding pocket [2]. In our current MD studies and OCM experiments we have used wild type and 2 different mutants of MUP to study the binding effects of the ligand IBM. The first mutant has an OH group removed from the binding site of MUP (i.e. tyrosine to phenylalanine (Y120F)). The second mutant has an extra OH group in the binding site (i.e. alanine to serine (A103S)). For all three systems the hydration and flexibility upon ligand binding has been analysed. The hydration analysis from MD reveal (from radial distribution curves and hydration density maps) there is a small density of water that remains even without the presence of the ligand for the WT MUP whereas a larger density of water remains in the binding cavity of the A103S hydrophilic MUP simulation. The results are based on the average structure generated from the 1 mus simulations. The Y120F MUP simulations reveal that there is no water molecules present in the binding cavity. However, as protein molecules are very dynamic in nature, water molecules are observed to hop in and out of the binding pockets for both mutant MUP (but not WT MUP) simulations over the 1 mus simulations. On the other hand the experimental QCM results reveal that on ligand binding no water loss is observed for Y120F mutant MUP whereas A103S and WT MUP have about 2 water molecules which are lost in the binding cavity. The flexibility results from the MD simulations reveal that WT MUP have some residues which increase in flexibility whilst other residues which decrease in flexibility on ligand binding. However, the Y120F hydrophobic MUP show an overall decrease in flexibility whereas the A103S MUP shows an overall increase in flexibility on ligand binding. In contrast the experimental OCM and AFM results reveal that there is an increase in flexibility on ligand binding to all 3 different types of MUP molecules. The experimental and the simulation data have shown a variation in results but it is to be noted that the results cannot be directly compared as the analytical experiments are a surface based techniques whereas the MD simulations do not involve a surface. However, the contrast observed between computer simulation and experiments has revealed important information on the ligand binding effects on MUP. [1] Bingham, R.J., J.B.C. Findlay, S.Y. Hsieh, A.P. Kalverda, A. Kjeliberg, C. Perazzolo, S.E.V. Phillips, K. Seshadri, C.H. Trinh, W. B. TurnbulI, G. Bodenhausen, and S.W. Homans. 2004. Thermodynamics of binding of 2-methoxy-3-lsopropylpyrazlne and 2- methoxy-3-lsobutylpyrazine to the major urinary protein. J. Am. Chem. Soc. 126:1675-1681. [2] Barratt, E., R.J. Bingham. D.J. Warner, C.A. Laughton, S.E.V. Phillips, and S.W. Homans. 2005. Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water. J. Am. Chem. Soc. 127:11827-11834

Ligand recognition by the major urinary protein

Molecular Dynamics (MD) and Quartz Crystal Microbalance (QCM) techniques can provide unique insights into what drives protein-ligand association. The major urinary protein (MUP) binds small ligands in a deeply buried hydrophobic pocket. Detailed calorimetric studies have shown that ligand binding is driven by enthalpic effects, not entropic effects [1]. Previous studies have shown that this is due to 'dewetting' of the binding site cavity even in the absence of ligands, and have also characterised the complex changes in molecular flexibility that accompany ligand binding-features that may be correlated with NMR data [2]. Recent MD revealed the hydration effects of apo-MUP and also shown where certain regions of MUP become more flexible upon ligand binding. They have also shown a water molecule remains close to the tyrosine in the binding pocket [2]. In our current MD studies and OCM experiments we have used wild type and 2 different mutants of MUP to study the binding effects of the ligand IBM. The first mutant has an OH group removed from the binding site of MUP (i.e. tyrosine to phenylalanine (Y120F)). The second mutant has an extra OH group in the binding site (i.e. alanine to serine (A103S)). For all three systems the hydration and flexibility upon ligand binding has been analysed. The hydration analysis from MD reveal (from radial distribution curves and hydration density maps) there is a small density of water that remains even without the presence of the ligand for the WT MUP whereas a larger density of water remains in the binding cavity of the A103S hydrophilic MUP simulation. The results are based on the average structure generated from the 1 mus simulations. The Y120F MUP simulations reveal that there is no water molecules present in the binding cavity. However, as protein molecules are very dynamic in nature, water molecules are observed to hop in and out of the binding pockets for both mutant MUP (but not WT MUP) simulations over the 1 mus simulations. On the other hand the experimental QCM results reveal that on ligand binding no water loss is observed for Y120F mutant MUP whereas A103S and WT MUP have about 2 water molecules which are lost in the binding cavity. The flexibility results from the MD simulations reveal that WT MUP have some residues which increase in flexibility whilst other residues which decrease in flexibility on ligand binding. However, the Y120F hydrophobic MUP show an overall decrease in flexibility whereas the A103S MUP shows an overall increase in flexibility on ligand binding. In contrast the experimental OCM and AFM results reveal that there is an increase in flexibility on ligand binding to all 3 different types of MUP molecules. The experimental and the simulation data have shown a variation in results but it is to be noted that the results cannot be directly compared as the analytical experiments are a surface based techniques whereas the MD simulations do not involve a surface. However, the contrast observed between computer simulation and experiments has revealed important information on the ligand binding effects on MUP. [1] Bingham, R.J., J.B.C. Findlay, S.Y. Hsieh, A.P. Kalverda, A. Kjeliberg, C. Perazzolo, S.E.V. Phillips, K. Seshadri, C.H. Trinh, W. B. TurnbulI, G. Bodenhausen, and S.W. Homans. 2004. Thermodynamics of binding of 2-methoxy-3-lsopropylpyrazlne and 2- methoxy-3-lsobutylpyrazine to the major urinary protein. J. Am. Chem. Soc. 126:1675-1681. [2] Barratt, E., R.J. Bingham. D.J. Warner, C.A. Laughton, S.E.V. Phillips, and S.W. Homans. 2005. Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water. J. Am. Chem. Soc. 127:11827-11834

Molecular Simulation Studies on the Prion Protein Variants: Insights into the Intriguing Effects of Mutations

Prion diseases, or transmissible spongiform encephalopathies (TSE), are a group of rare fatal neurodegenerative maladies that affect humans and animals. The fundamental breakthrough in TSE research was the discovery of the "prion"\u23afproteinaceous infectious particle\u23af and the verification of the \u201cprotein-only\u201d hypothesis, which states that prions could self-propagate by converting the cellular prion protein (PrPC) into the scrapie form, PrPSc (or prions), and lead to neurodegeneration without using any nucleic acids. The concept of prions may unify neurodegenerative diseases under a common pathogenic mechanism. Indeed, growing evidence shows that TSE may share similar pathogenesis with common neurodegenerative syndromes such as Alzheimer\u2019s disease and Parkinson\u2019s disease, for which there are currently no cure. Today, PrP is one of the most studied models for protein misfolding mechanism and TSE serve as an excellent model for studying many other neurodegenerative diseases. Understanding the molecular mechanism of the PrP misfolding process may profoundly influence the development of diagnostics and effective therapies for neurodegenerative diseases in general. Investigating human (Hu) PrP TSE-linked mutations (more than 50 currently identified mutations, linked to ~15% of the cases) may be very instrumental in this respect, as it can provide hints on the molecular basis of the PrPC\u2192PrPSc conversion. These mutations cause spontaneous TSE, which are likely due to modifications in the native structure of PrPC. They are located all over the structure. Polymorphisms (i.e. non-pathogenic, naturally occurring mutations) in the PrP gene have been found to influence the etiology and neuropathology of the disease in both humans and sheep. In transgenic (Tg) mice, artificial mutations can determine the susceptibility to the infection of different prion strains. Intriguingly, mouse (Mo) PrP containing artificial mutations (denoted MoPrP chimera, hereinafter) have very different effects in vitro: some MoPrP chimera were found to resist PrPSc infection, whereas some others did not; some of the resistant MoPrP chimeras even exhibited a protective effect (known as the dominant-negative effect) over the co-expressed endogenous wild-type (WT) MoPrPC. Most mutations are located in the folded globular domain (GD) while fewer are located in the intrinsically disordered N-terminal domain (N-term). The N-term of PrPC has been suggested to serve multiple functions in vivo, which likely relies on the structural flexibility of this domain. Therefore, characterizing the structural features of the N-term is central for investigating not only the mutations in this domain, but also the physiological role of the N-term. Based on previous studies in our lab, in this thesis we first applied molecular dynamics simulations to studying the impact of all the known Hu TSE-linked mutations in HuPrPC GD. We next applied the same approach to study the GD structure of MoPrP chimeras which contain one or two residues from Hu or sheep PrP sequence. By studying these PrP variants, we aim to identify the structural determinants of the mutants that may play a role in the PrPC\u2192PrPSc conversion. Our calculations discovered that these mutants exhibit different structural features from those of the WT PrP GD mainly in two common regions that are likely the \u201chot spots\u201d in the protein misfolding process. These features can be classified into different types that are correlated to the types of mutants (i.e. pathogenic, resistant or dominant-negative), thus hinting to the molecular mechanisms of PrPSc formation and propagation. We have then predicted the structure of the entire PrP N-term and the impact of the Hu TSE-linked mutations in this domain using a novel Monte Carlo-based simulation approach, PROFASI. PROFASI has already shown to provide structural predictions in a disordered protein such as \u3b1-synuclein. Our results are consistent with available experimental data and therefore firmly allow us to provide the first overview on the structural determinants of all Hu TSE-linked mutations in PrP

Development and Application of a Novel Enhanced Sampling Method and Bayesian Analysis for Characterizing Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) are a class of proteins with wide-rangingsignificance in signaling and disease that do not adopt a dominant folded structure asmonomers. Rather, the structures of IDPs in solution are best described as ensembles ofconformational states that may range from being fully random coil to partially ordered.This structural plasticity of IDPs is theorized to facilitate regulation of their interactionwith other species, as in signal transduction or aggregation of IDPs into ordered fibrils.Characterizing the structural ensembles of IDPs in the free, solvated state is key tounderstanding the mechanisms of these interactions, and correspondingly the role an IDPspecies plays in signaling or disease.The rapid interconversion between conformational states, however, complicates theexperimental study of IDPs because most experimental signals report highly averagedinformation. Computational modeling with validation through comparison to experimenthas therefore been a main approach to characterizing IDP structure and dynamics. Thefocus of my dissertation is on the development of new methods for computational study ofIDPs, facilitating better and less expensive de novo generation of IDP structural ensemblesand improving the metrics used to evaluate the degree of agreement between a simulatedensemble and a set of experimental data.Despite vast improvements in computational power and efficiency, moleculardynamics (MD) simulations of IDPs for generating conformational ensembles are stilllimited by the expense of calculations. In Chapter 2 I present the development of a newenhanced sampling method – temperature cool walking (TCW) – and comparison of itsperformance against a standard method – temperature replica exchange (TREx). The TCWmethod accelerates the rate of convergence to the equilibrium conformational ensemblewith increased sampling acceleration relative to TREx at greatly reduced computationalcost.The second major limitation in MD is the accuracy of the force field. Most classicalfixed charge force fields were parameterized using data from folded proteins, and havebeen thought to be biased to overly collapsed and structured conformations. This hasmotivated the development of IDP-tailored force fields that sample greater disorder, at thepotential expense of the ability to model stabilizing interactions between an IDP and itsbinding partners. In Chapter 3, I assess to what degree the shortcomings assigned tostandard force fields may be due to insufficient sampling by characterizing theperformance of standard and newly modified force fields on the Alzheimer’s peptideamyloid-β using both TREx and TCW. We find that with improved sampling, standard andmodified force fields produce similar structural ensembles, suggesting that both areappropriate for simulation of the disordered state. In Chapter 4 I present preliminaryresults building off of this work by characterizing the performance of a polarizable forcefield modeling a synthetic peptide that demonstrates complete loss of helical content withincreasing temperature. Inclusion of polarization effects has been thought to be key foraccurate modeling of such multicomponent systems, especially when there is a shift in theelectrostatic environment as is the case for the unfolding peptide. Our early results, whilelimited by current lack of convergence for tests using the polarizable force field andneeding further confirmation, match that expectation by finding early evidence of greaterresponse to temperature by the polarizable force field than fixed charge comparators.The last work presented here is in the development of new methods for calculatingthe degree of agreement between a simulated IDP ensemble and experimental data. Backcalculationof experimental data from structure can be very imprecise, motivating thedevelopment in Chapter 5 of scoring formalisms that account for variable uncertainties inboth back-calculation and experiment for diverse experimental data types. In summary, themethods described in this dissertation seek to improve computational study of IDPs byfacilitating better, less expensive generation of IDP ensembles and producing moreinformative metrics for evaluating their agreement with experiment