ABodyBuilder: Automated antibody structure prediction with data–driven accuracy estimation

Computational modeling of antibody structures plays a critical role in therapeutic antibody design. Several antibody modeling pipelines exist, but no freely available methods currently model nanobodies, provide estimates of expected model accuracy, or highlight potential issues with the antibody's experimental development. Here, we describe our automated antibody modeling pipeline, ABodyBuilder, designed to overcome these issues. The algorithm itself follows the standard 4 steps of template selection, orientation prediction, complementarity-determining region (CDR) loop modeling, and side chain prediction. ABodyBuilder then annotates the ‘confidence’ of the model as a probability that a component of the antibody (e.g., CDRL3 loop) will be modeled within a root–mean square deviation threshold. It also flags structural motifs on the model that are known to cause issues during in vitro development. ABodyBuilder was tested on 4 separate datasets, including the 11 antibodies from the Antibody Modeling Assessment–II competition. ABodyBuilder builds models that are of similar quality to other methodologies, with sub–Angstrom predictions for the ‘canonical’ CDR loops. Its ability to model nanobodies, and rapidly generate models (～30 seconds per model) widens its potential usage. ABodyBuilder can also help users in decision–making for the development of novel antibodies because it provides model confidence and potential sequence liabilities. ABodyBuilder is freely available at http://opig.stats.ox.ac.uk/webapps/abodybuilder.     

Molecular dynamics simulation of lactate dehydrogenase adsorption onto pristine and carboxylic-functionalized graphene

ABSTRACT

Lactate dehydrogenase (LDH) is a tetrameric enzyme which is composed of two subunits known as LDHA and LDHB, which are encoded by the LDHA and LDHB genes respectively. LDH catalyses the last step in anaerobic glycolysis through the reversible conversion of pyruvate to lactate via coupled oxidation of NADH cofactor. The LDHA plays an important regulatory role in anaerobic glycolysis, by catalysing the final step of the process. Therefore, it is likely that increases in the expression level of LDHA in cancer cells could facilitate the efficiency of anaerobic glycolysis. Measuring the level of serum LDHA is a key step in the diagnosis of many cancer types. In this study, the adsorption, stability, and dynamics of LDHA on the surface of pristine graphene (PG) and carboxylated graphene (COOH-Graphene) were investigated using its molecular dynamics simulation. Variations in root mean square deviation, root mean square fluctuation, solvent accessible surface area and adsorption energy of the LDHA during the simulation were calculated to analyse the effect of PG and COOH-Graphene on the overall conformation of LDHA. Results showed that the adsorption of LDHA on COOH-Graphene is mostly mediated by electrostatic interactions, whereas on the PG, both Van der Waals and π-π interactions are prominent.     

Delineating the conformational dynamics of intermediate structures on the unfolding pathway of β-lactoglobulin in aqueous urea and dimethyl sulfoxide

Abstract

The funnel shaped energy landscape model of the protein folding suggests that progression of folding proceeds through multiple pathways, having the multiple intermediates which leads to multidimensional free-energy surface. Herein, we applied all-atom MD simulation to conduct a comparative study on the structure of β-lactoglobulin (β-LgA) in aqueous mixture of 8?M urea and 8?M dimethyl sulfoxide (DMSO), at different temperatures. The cumulative results of multiple simulations suggest a common unfolding pathway of β-LgA, occurred through the stable and meta-stable intermediates (I), in both urea and DMSO. However, the free-energy landscape (FEL) analyses show that the structural transitions of I-states are energetically different. In urea, FEL shows distinct ensemble of intermediates, I1 and I2, separated by the energy barrier of ～3.0?kcal mol^?1. Similarly, we find the population of two distinct I1 and I2 states in DMSO, however, the I1 appeared transiently around ～30–35?ns and is short-lived. But, the I2 ensemble is observed structurally compact and long-lived (～50–150?ns) as compared to unfolding in urea. Furthermore, the I1 and I2 are separated through a high energy barrier of ～6.0?kcal mol^?1. Thus, our results provide the structural insights of intermediates which essentially bear the signature of a different unfolding pathway of β-LgA in urea and DMSO.

Abbreviations β-LgA β-lactoglobulin

DMSO dimethyl sulfoxide

FEL free-energy landscape

GdmCl guanidinium chloride

I intermediate state

MG molten globule state

PME particle mesh Ewald

Q fraction of native contacts

RMSD root mean square deviation

RMSF root mean square fluctuation

R_g radius of gyration

SASA solvent Accessible Surface Area

scSASA the side chain SASA

Trp tryptophan

Communicated by Ramaswamy H. Sarma     

CDR-H3 loop ensemble in solution – conformational selection upon antibody binding

ABSTRACT

We analyzed pairs of protein-binding, peptide-binding and hapten-binding antibodies crystallized as complex and in the absence of the antigen with and without conformational differences upon binding in the complementarity-determining region (CDR)-H3 loop. Here, we introduce a molecular dynamics-based approach to capture a diverse conformational ensemble of the CDR-H3 loop in solution. The results clearly indicate that the inherently flexible CDR-H3 loop indeed needs to be characterized as a conformational ensemble. The conformational changes of the CDR-H3 loop in all antibodies investigated follow the paradigm of conformation selection, because we observe the experimentally determined binding competent conformation without the presence of the antigen within the ensemble of pre-existing conformational states in solution before binding. We also demonstrate for several examples that the conformation observed in the antibody crystal structure without antigen present is actually selected to bind the carboxyterminal tail region of the antigen-binding fragment (Fab). Thus, special care must be taken when characterizing antibody CDR-H3 loops by Fab X-ray structures, and the possibility that pre-existing conformations are present should always be considered.     

抗体片段在铂纳米粒子表面的构象重构

目的 最近在金纳米粒子（AuNPs）表面重构抗体片段的天然构象和功能的研究表明分子构象工程的可行性。本质上,分子构象工程就是要像蛋白质折叠一样,通过精确控制柔性非功能分子的构象使其产生新功能。本文在铂纳米粒子（PtNPs）表面重构抗体互补决定簇区（CDR）片段的天然构象和功能,旨在探索分子构象工程的普适性及揭示蛋白质结构-功能机制。方法 本文将抗溶菌酶抗体（cAB-lys3）中的CDR3片段（在单独存在时没有稳定构象和功能）通过两个Pt-S键偶联到PtNPs表面。CDR片段的天然构象和功能的恢复通过它对溶菌酶活性的抑制来表征。结果 通过多肽密度优化和表面聚乙二醇修饰,制得基于PtNPs的抗溶菌酶人工抗体（简称铂抗体）。溶菌酶活性测试结果表明,铂抗体可以特异性结合溶菌酶并显著抑制其活性。结论 本文第一次在PtNPs表面重构了蛋白质片段的天然构象并恢复了其功能,证明分子构象工程可作为一种通用方法制备基于纳米粒子的人工蛋白质。     

Applications of nanobodies in plant science and biotechnology

Key message

Present review summarizes the current applications of nanobodies in plant science and biotechnology, including plant expression of nanobodies, plant biotechnological applications, nanobody-based immunodetection, and nanobody-mediated resistance against plant pathogens.

Abstract

Nanobodies (Nbs) are variable domains of heavy chain-only antibodies (HCAbs) isolated from camelids. In spite of their single domain structure, nanobodies display many unique features, such as small size, high stability, and cryptic epitopes accessibility, which make them ideal for sophisticated applications in plants and animals. In this review, we summarize the current applications of nanobodies in plant science and biotechnology, focusing on nanobody expression in plants, plant biotechnological applications, determination of plant toxins and pathogens, and nanobody-mediated resistance against plant pathogens. Prospects and challenges of nanobody applications in plants are also discussed.

     

Ab initio structure prediction of the antibody hypervariable H3 loop

Antibodies have the capability of binding a wide range of antigens due to the diversity of the six loops constituting the complementarity determining region (CDR). Among the six loops, the H3 loop is the most diverse in structure, length, and sequence identity. Prediction of the three‐dimensional structures of antibodies, especially the CDR loops, is an important step in the computational design and engineering of novel antibodies for improved affinity and specificity. Although it has been demonstrated that the conformation of the five non‐H3 loops can be accurately predicted by comparing their sequences against databases of canonical loop conformations, no such connection has been established for H3 loops. In this work, we present the results for ab initio structure prediction of the H3 loop using conformational sampling and energy calculations with the program Prime on a dataset of 53 loops ranging in length from 4 to 22 residues. When the prediction is performed in the crystal environment and including symmetry mates, the median backbone root mean square deviation (RMSD) is 0.5 Å to the crystal structure, with 91% of cases having an RMSD of less than 2.0 Å. When the prediction is performed in a noncrystallographic environment, where the scaffold is constructed by swapping the H3 loops between homologous antibodies, 70% of cases have an RMSD below 2.0 Å. These results show promise for ab initio loop predictions applied to modeling of antibodies. © 2012 Wiley Periodicals, Inc.     

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10 Å from the active site, while epitope β is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a β epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.     

Modulation of p53 N-terminal transactivation domain 2 conformation ensemble and kinetics by phosphorylation

Abstract

Phosphorylation of protein is critical for various cell processes, which preferentially happens in intrinsically disordered proteins (IDPs). How phosphorylation modulates structural ensemble of disordered peptide remains largely unexplored. Here, using replica exchange molecular dynamics (REMD) and Markov state model (MSM), the conformational distribution and kinetics of p53 N-terminal transactivation domain (TAD) 2 as well as its dual-site phosphorylated form (pSer46, pThr55) were simulated. It reveals that the dual phosphorylation does not change overall size and secondary structure element fraction, while a change in the distribution of hydrogen bonds induces slightly more pre-existing bound helical conformations. MSM analysis indicates that the dual phosphorylation accelerates conformation exchange between disordered and order-like states in target-binding region. It suggests that p53 TAD2 after phosphorylation would be more apt to bind to both the human p62 pleckstrin homology (PH) domain and the yeast tfb1?PH domain through different binding mechanism, where experimentally it exhibits an extended and α-helix conformation, respectively, with increased binding strength in both complexes. Our study implies except binding interface, both conformation ensemble and kinetics should be considered for the effects of phosphorylation on IDPs. Abbreviations IDPs intrinsically disordered proteins

REMD replica exchange molecular dynamics

MSM Markov state model

TAD transactivation domain

PH pleckstrin homology

PRR proline-rich region

DBD DNA-binding domain

TET Tetramerization domain

REG regulatory domain

MD molecular dynamics

PME particle-mesh Ewald

TICA time-lagged independent component analysis

CK Chapman–Kolmogorov

GMRQ generalized matrix Rayleigh quotient

SARW self-avoiding random walk

KID kinase-inducible domain

MFPT mean first passage time

DSSP definition of secondary structure of proteins

RMSD root mean square deviation

R_g radius of gyration

R_ee end to end distance

Communicated by Ramaswamy H. Sarma     

Discrete analyses of protein dynamics

Abstract

Protein structures are highly dynamic macromolecules. This dynamics is often analysed through experimental and/or computational methods only for an isolated or a limited number of proteins. Here, we explore large-scale protein dynamics simulation to observe dynamics of local protein conformations using different perspectives. We analysed molecular dynamics to investigate protein flexibility locally, using classical approaches such as RMSf, solvent accessibility, but also innovative approaches such as local entropy. First, we focussed on classical secondary structures and analysed specifically how β-strand, β–turns, and bends evolve during molecular simulations. We underlined interesting specific bias between β–turns and bends, which are considered as the same category, while their dynamics show differences. Second, we used a structural alphabet that is able to approximate every part of the protein structures conformations, namely protein blocks (PBs) to analyse (i) how each initial local protein conformations evolve during dynamics and (ii) if some exchange can exist among these PBs. Interestingly, the results are largely complex than simple regular/rigid and coil/flexible exchange. Abbreviations N_eq number of equivalent

PB Protein Blocks

PDB Protein DataBank

RMSf root mean square fluctuations

Communicated by Ramaswamy H. Sarma     

Population genetics of Himalayan balsam (Impatiens glandulifera): comparison of native and introduced populations

Background: Invasive species can interfere in the structure and functioning of ecosystems. Better understanding of the evolution of such species will be useful when planning their management and eradication.

Aims: We aimed to compare patterns of genetic variability in Impatiens glandulifera in native and introduced regions.

Methods: We used native samples from India and Pakistan, and non-native samples from Canada, Finland and the UK. Genetic analyses included genotyping using 10 microsatellite markers and sequencing of the nuclear ITS region.

Results: Mean allele numbers from native and introduced samples were even, 8.8 and 8.5, respectively, while expected heterozygosities were higher in native samples (mean 0.738) than in non-native samples (mean 0.477). Hardy–Weinberg equilibrium testing indicated significant heterozygote deficiencies at 70% of the loci. Inbreeding coefficients were high in both native and introduced regions (range 0.201–0.726). STRUCTURE analyses showed that native samples from India and Pakistan possessed similar clustering patterns while non-native samples from the UK and Canada resembled each other. One of the four Finnish populations had a similar pattern with the UK and Canadian populations, while the rest showed similarly unique genetic compositions. ITS sequencing indicated in Pakistani samples two polymorphic sites not found in Indian samples but present in some samples from Canada, Finland and the UK.

Conclusions: Distinct population genetic patterns indicate that human-mediated dispersal is important in I. glandulifera.     

A single residue exchange between two HLA-B27 alleles triggers increased peptide flexibility

For more than 30 years, human leukocyte antigen B27 (HLA-B27) has been known to be closely related to the autoimmune disease ankylosing spondylitis, yet little is known about the molecular mechanisms of pathogenesis. Crystal structures of two closely related, but differently disease-associated, subtypes (B*2705 and B*2709) also did not resolve this situation as they revealed the bound nonapeptide in essentially identical conformations. As the peptide is part of putative binding epitopes for the T cell receptor, we performed molecular dynamics simulations to gain deeper insight into the dynamic behaviour of HLA-B27 molecules. We find increased flexibility of the peptide in the binding groove of subtype B*2709 due to weaker interactions in the F pocket. Possible implications of this flexibility for T cell recognition and signalling are discussed.Abbreviations ₂m ₂-microglobulin - AS ankylosing spondylitis - CDR complementarity determining region - HC heavy chain - HLA human leukocyte antigen - MD molecular dynamics - MHC major histocompatibility complex - pMHC peptide-loaded MHC - RMSD root mean square deviation - RMSF root mean square fluctuation - TCR T cell receptor An erratum to this article can be found at     

Structural dynamics behind variants in pyrazinamidase and pyrazinamide resistance

Abstract

Pyrazinamide (PZA) is an important component of first-line anti-tuberculosis (anti-TB) drugs. The anti-TB agent is activated into an active form, pyrazinoic acid (POA), by Mycobacterium tuberculosis (MTB) pncA gene encoding pyrazinamidase (PZase). The major cause of PZA-resistance has been associated with mutations in the pncA gene. We have detected several novel mutations including V131F, Q141P, R154T, A170P, and V180F (GeneBank Accession No. MH461111) in the pncA gene of PZA-resistant isolates during PZA drug susceptibility testing followed by pncA gene sequencing. Here, we investigated molecular mechanism of PZA-resistance by comparing the results of experimental and molecular dynamics. The mutants (MTs) and wild type (WT) PZase structures in apo and complex with PZA were subjected to molecular dynamic simulations (MD) at the 40?ns. Multiple factors, including root mean square deviations (RMSD), binding pocket, total energy, dynamic cross correlation, and root mean square fluctuations (RMSF) of MTs and WT were compared. The MTs attained a high deviation and fluctuation compared to WT. Binding pocket volumes of the MTs, were found, lower than the WT, and the docking scores were high than WT while shape complementarity scores were lower than that of the WT. Residual motion in MTs are seemed to be dominant in anti-correlated motion. Mutations at locations, V131F, Q141P, R154T, A170P, and V180F, might be involved in the structural changes, possibly affecting the catalytic property of PZase to convert PZA into POA. Our study provides useful information that will enhance the understanding for better management of TB. Abbreviations DST drug susceptibility testing

Δelec electrostatic energy

LJ Lowenstein–Jensen medium

MGIT mycobacterium growth indicator tubes

MTs mutants

MD molecular dynamic simulations

MTB Mycobacterium tuberculosis

NALC–NaOH N-acetyl-l-cysteine–sodium hydroxide

NIH National Institutes of Health

NPT amount of substance (N), pressure (P) temperature (T)

NVT moles (N), volume (V) temperature (T)

PZase pyrazinamidase

Δps polar solvation energy

PTRL Provincial Tuberculosis Reference Laboratory

RMSD root mean square deviations

RMSF root mean square fluctuations

ΔSASA solvent accessible surface area energy

TB tuberculosis

GTotal total binding free energy

ΔvdW Van der Waals energy

WT wild type

Communicated by Ramaswamy H. Sarma     

Ab initio folding simulation of the Trp-cage mini-protein approaches NMR resolution

Here, we report a 100 ns molecular dynamics simulation of the folding process of a recently designed autonomous-folding mini-protein designated as tc5b with a new AMBER force field parameter set developed based on condensed-phase quantum mechanical calculations and a Generalized Born continuum solvent model. Starting from its fully extended conformation, our simulation has produced a final structure resembling that of NMR native structure to within 1A main-chain root mean square deviation. Remarkably, the simulated structure stayed in the native state for most part of the simulation after it reached the state. Of greater significance is that our simulation has not only reached the correct main-chain conformation, but also a very high degree of accuracy in side-chain packing conformation. This feat has traditionally been a challenge for ab initio simulation studies. In addition to characterization of the trajectory, comparison of our results to experimental data is also presented. Analysis of the trajectory suggests that the rate-limiting step of folding of this mini-protein is the packing of the Trp side-chain.     

Model and Molecular Dynamic Simulations of Active and Inactive Endo-β-1,4-Mannanase in Tomato Fruit

Endo-β-mannanase is a hemicellulase that is present in tomato fruit, and plays a role in its ripening. This enzyme protein is detectable in the cultivar Walter, but it is inactive due to the absence of the terminal four amino acids from its carboxyl-end. To elucidate why this deletion eliminates the activity of endo-β-mannanase, a molecular dynamics (MD) study was conducted on the conformation of the enzyme at normal and elevated temperatures. The root mean square deviations, root mean square fluctuations per residue, and secondary structural evolution during MD simulations were analyzed. Differences in stability and dynamics between the active and inactive endo-β-mannanases were documented; the inactive form has a lower stability than the active one. The loss of key amino acids from the C-terminal end of the protein indirectly affects the conformation of the catalytic Glu318 and stability of active site because of interactions between residues at the C-terminus and the rest of protein.     

Germination responses of native and invasive Cerrado grasses to simulated fire temperatures

Background: Fire is an important ecological factor in the Cerrado (Brazilian savanna). However, comparative studies on the effect of high temperatures experienced during fires on seed germination of native and invasive grass species are few.

Aims: To assess germination responses to simulated fire temperatures by seeds of invasive and native Cerrado grasses.

Methods: Heat-shock treatments (50 °C, 70 °C, 90 °C, 110 °C, 130 °C or 150 °C) were applied to seeds of 10 species of native and invasive grasses. For each temperature, the seeds were heated in a dry-air flow for 2 or 5 min. This combination of temperatures and exposure times simulated the soil conditions during typical Cerrado fires.

Results: Temperature treatment was significantly related to germination, and the effect varied according to species. Heat shock did not increase germination in either the native or the invasive species. Exposure time was important for only two species, and four species showed a significant increase in mean germination time.

Conclusions: Species showed different tolerances to high temperatures. It was not possible to differentiate the native and invasive grasses only by their tolerance to high temperatures, suggesting that fire alone may not be an efficient management tool to control the invasive species studied here.     

Multi‐constraint computational design suggests that native sequences of germline antibody H3 loops are nearly optimal for conformational flexibility

The limited size of the germline antibody repertoire has to recognize a far larger number of potential antigens. The ability of a single antibody to bind multiple ligands due to conformational flexibility in the antigen‐binding site can significantly enlarge the repertoire. Among the six complementarity determining regions (CDRs) that generally comprise the binding site, the CDR H3 loop is particularly variable. Computational protein design studies showed that predicted low energy sequences compatible with a given backbone structure often have considerable similarity to the corresponding native sequences of naturally occurring proteins, indicating that native protein sequences are close to optimal for their structures. Here, we take a step forward to determine whether conformational flexibility, believed to play a key functional role in germline antibodies, is also central in shaping their native sequence. In particular, we use a multi‐constraint computational design strategy, along with the Rosetta scoring function, to propose that the native sequences of CDR H3 loops from germline antibodies are nearly optimal for conformational flexibility. Moreover, we find that antibody maturation may lead to sequences with a higher degree of optimization for a single conformation, while disfavoring sequences that are intrinsically flexible. In addition, this computational strategy allows us to predict mutations in the CDR H3 loop to stabilize the antigen‐bound conformation, a computational mimic of affinity maturation, that may increase antigen binding affinity by preorganizing the antigen binding loop. In vivo affinity maturation data are consistent with our predictions. The method described here can be useful to design antibodies with higher selectivity and affinity by reducing conformational diversity. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Identification of therapeutic peptide scaffold from tritrpticin family for urinary tract infections using in silico techniques

Abstract

Antimicrobial peptides (AMPs) like tritrpticins, exhibit non-specific membrane lysis of gram-negative bacteria and can replace antibiotics, combating multi-drug resistance observed in UTI patients. Tritrpticins designated – NT, T1, T2, T3, T5, T7 and T8, were computationally investigated by interaction with Escherichia coli membrane model, mammalian cell toxicity and structural stability to identify a potential drug scaffold for UTI. Initially T3 was eliminated due to low interaction with Escherichia coli membrane model, based on its computed solvation energy. Further, negative support vector machine (SVM) scores revealed non-toxicity of T1, T2, T5, T7 and T8. Finally, at 310?K and varying pH 4.5–9.0, T5 exhibited highest structural stability based on its highest consistency of hydrogen bonds (H-bonds), root mean square deviation (RMSD) and secondary structure profiles along with its lowest conformational free energy. Overall, T5 could be considered a promising peptide drug scaffold to combat UTI.

ABBREVIATIONS AMP antimicrobial peptide

PBEQ Poisson Boltzmann equation

H-bonds hydrogen bonds

MIC minimum inhibitory concentration

LD50 lethal dose, 50%

RMSD root mean square deviation

SVM support vector machine

UTI urinary tract infection

Communicated by Ramaswamy H. Sarma     

Role of ELA region in auto-activation of mutant KIT receptor: a molecular dynamics simulation insight

KIT receptor is the prime target in gastrointestinal stromal tumor (GISTs) therapy. Second generation inhibitor, Sunitinib, binds to an inactivated conformation of KIT receptor and stabilizes it in order to prevent tumor formation. Here, we investigated the dynamic behavior of wild type and mutant D816H KIT receptor, and emphasized the extended A-loop (EAL) region (805–850) by conducting molecular dynamics simulation (～100?ns). We analyzed different properties such as root mean square cutoff or deviation, root mean square fluctuation, radius of gyration, solvent-accessible surface area, hydrogen bonding network analysis, and essential dynamics. Apart from this, clustering and cross-correlation matrix approach was used to explore the conformational space of the wild type and mutant EAL region of KIT receptor. Molecular dynamics analysis indicated that mutation (D816H) was able to alter intramolecular hydrogen bonding pattern and affected the structural flexibility of EAL region. Moreover, flexible secondary elements, specially, coil and turns were dominated in EAL region of mutant KIT receptor during simulation. This phenomenon increased the movement of EAL region which in turn helped in shifting the equilibrium towards the active kinase conformation. Our atomic investigation of mutant KIT receptor which emphasized on EAL region provided a better insight into the understanding of Sunitinib resistance mechanism of KIT receptor and would help to discover new therapeutics for KIT-based resistant tumor cells in GIST therapy.     

Conformation analysis of a surface loop that controls active site access in the GH11 xylanase A from Bacillus subtilis

Xylanases (EC 3.2.1.8 endo-1,4-glycosyl hydrolase) catalyze the hydrolysis of xylan, an abundant hemicellulose of plant cell walls. Access to the catalytic site of GH11 xylanases is regulated by movement of a short β-hairpin, the so-called thumb region, which can adopt open or closed conformations. A crystallographic study has shown that the D11F/R122D mutant of the GH11 xylanase A from Bacillus subtilis (BsXA) displays a stable “open” conformation, and here we report a molecular dynamics simulation study comparing this mutant with the native enzyme over a range of temperatures. The mutant open conformation was stable at 300 and 328 K, however it showed a transition to the closed state at 338 K. Analysis of dihedral angles identified thumb region residues Y113 and T123 as key hinge points which determine the open-closed transition at 338 K. Although the D11F/R122D mutations result in a reduction in local inter-intramolecular hydrogen bonding, the global energies of the open and closed conformations in the native enzyme are equivalent, suggesting that the two conformations are equally accessible. These results indicate that the thumb region shows a broader degree of energetically permissible conformations which regulate the access to the active site region. The R122D mutation contributes to the stability of the open conformation, but is not essential for thumb dynamics, i.e., the wild type enzyme can also adapt to the open conformation.