The role of Asn14 in the stability and conformation of the reactive-site loop of winged bean chymotrypsin inhibitor: crystal structures of two point mutants Asn14{->}Lys and Asn14{->}Asp

A double-headed chymotrypsin inhibitor, WCI, from winged beanseeds was cloned for structural and biochemical studies. Theinhibitor was subjected to two point mutations at a conservedposition, Asn14. This residue, known to have a pivotal rolein stabilizing the first reactive-site loop (Gln63–Phe68)of the inhibitor, is highly conserved in the sequences of theother members of Kunitz (STI) family as well as in the sequencesof Kazal family of serine protease inhibitors. The mutants,N14K and N14D, were subjected to biochemical assay and theircharacteristics were compared with those of the recombinantinhibitor (rWCI). Crystallographic studies of the recombinantand the mutant proteins are discussed. These studies were primarilyaimed at understanding the importance of the protein scaffoldingtowards the conformational rigidity of the reactive-site loop.Our analysis reveals that, as the Lys14 side chain takes anunusual fold in N14K and the Asp14 side chain in N14D interactswith the loop residues by water-mediated hydrogen bonds, thecanonical conformation of the loop has remained effectivelyintact in both the mutant structures. However, minor alterationssuch as a 2-fold increase in the inhibitory affinity towardsthe cognate enzyme were observed.     

A Comparative Study to Decipher the Structural and Dynamics Determinants Underlying the Activity and Thermal Stability of GH-11 Xylanases

With the growing need for renewable sources of energy, the interest for enzymes capable of biomass degradation has been increasing. In this paper, we consider two different xylanases from the GH-11 family: the particularly active GH-11 xylanase from Neocallimastix patriciarum, NpXyn11A, and the hyper-thermostable mutant of the environmentally isolated GH-11 xylanase, EvXyn11^TS. Our aim is to identify the molecular determinants underlying the enhanced capacities of these two enzymes to ultimately graft the abilities of one on the other. Molecular dynamics simulations of the respective free-enzymes and enzyme–xylohexaose complexes were carried out at temperatures of 300, 340, and 500 K. An in-depth analysis of these MD simulations showed how differences in dynamics influence the activity and stability of these two enzymes and allowed us to study and understand in greater depth the molecular and structural basis of these two systems. In light of the results presented in this paper, the thumb region and the larger substrate binding cleft of NpXyn11A seem to play a major role on the activity of this enzyme. Its lower thermal stability may instead be caused by the higher flexibility of certain regions located further from the active site. Regions such as the N-ter, the loops located in the fingers region, the palm loop, and the helix loop seem to be less stable than in the hyper-thermostable EvXyn11^TS. By identifying molecular regions that are critical for the stability of these enzymes, this study allowed us to identify promising targets for engineering GH-11 xylanases. Eventually, we identify NpXyn11A as the ideal host for grafting the thermostabilizing traits of EvXyn11^TS.     

A salt-bridge controlled by ligand binding modulates the hydrolysis reaction in a GH5 endoglucanase

Cellulases, distributed in at least 15 families of glycoside hydrolases, will play a key role in biomass conversion and renewable energy challenges of the future. Cel5B from Clostridium thermocellum is a β-1,4-endoglucanase and a member of family 5 of glycoside hydrolases (GH5) and is characterized by an (α/β)(8) barrel structure. In contrast to other retaining enzymes, in which the catalytic carboxylate groups (glutamate or aspartate) are positioned ≈ 5.5 ? apart to facilitate nucleophilic attack on the anomeric carbon of the sugar substrate, these two residues in Cel5B are positioned ≈ 10 ? from each other in the unliganded wild-type structure. The structure of the enzyme solved in complex with a cleavage product (cellobiose) revealed ligand-induced conformational changes in the loop carrying Glu140 (proton donor). The reorientation of Glu140 in the complex reduces the separation of the catalytic glutamate residues to 4.3 ?. In this study, we took advantage of conventional and steered molecular dynamics (MD) simulations along with in silico and in vitro mutagenesis to investigate the ligand-induced changes of the enzyme and interactions involved in preservation of Cel5B conformations in the presence and absence of substrate. We determined that the variation in separation of catalytic glutamates in the absence and presence of substrate is due to the different protonation states of the proton donor glutamate that is largely governed by conformational changes in the β3α3 loop. In the absence of substrate, the conformation of Cel5B is preserved by an electrostatic interaction between deprotonated Glu140 and protonated His91. The ion pair is interrupted upon the binding of substrate, and the positional displacement of the β3α3 loop allows Glu140 to become oriented within the active site in a less hydrophilic microenvironment that assists in Glu140 protonation.     

Molecular Modelling—an Enabling Technology for Chemical Engineers

This article briefly describes the basic concepts involved in the two most commonly used molecular modelling methods—molecular dynamics (MD) and Monte Carlo (MC). The methods are particularly useful for studying structures at the length scale of nanometre. Two examples (both are on the study of the miscibility of polyolefin blends) are used to illustrate the techniques. It is demonstrated that it is the nano‐scaled structures formed by the segments of the constituent polyolefins that prevent them from mixing with each other. The examples also show that selection of specific method (MD or MC) depends on the nature of the problem in hand. In general, MC is more efficient than MD in terms of generating equilibrated structure while MD can provide information about the dynamics of a system. This is simply because MD requires the solution of equations of motion (a set of second order differential equations) while MC does not. Nonetheless, both methods need a reasonably accurate force field.     

The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel

Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli. Each subunit contains 16 beta-strands forming a transmembrane beta-barrel whose pore is constricted by the third extracellular loop (L3). We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation targets a key component of a putative hydrogen bond network linking the L3 loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mutations target putative salt bridges at the root of the L3 loop. We purified the outer membrane fractions obtained from each mutant and reconstituted them in liposomes suitable for electrophysiology. Patch clamp experiments showed that the frequency of spontaneous transitions between open and closed states is increased in the D315A, D118Q and R92Q mutants but unchanged in the R174Q mutant. These transitions are not driven by transmembrane voltage changes and represent the thermal oscillations between functionally distinct conformations. The asymmetric voltage-dependent inactivation of the channels is not affected by the mutations, however, suggesting different molecular mechanisms for the spontaneous and voltage- dependent gating processes. We propose that the positioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role in determining the frequency of spontaneous channel gating.      

Exploring the potential of the monobody scaffold: effects of loop elongation on the stability of a fibronectin type III domain

The tenth fibronectin type III domain of human fibronectin (FNfn10)is a small, monomeric ß-sandwich protein, similarto the immunoglobulins. We have developed small antibody mimics,‘monobodies’, using FNfn10 as a scaffold. We initiallyaltered two loops of FNfn10 that are structurally equivalentto two of the hypervariable loops of the immunoglobulin domain.In order to assess the possibility of utilizing other loopsin FNfn10 for target binding, we determined the effects of theelongation of each loop on the conformational stability of FNfn10.We found that all six loops of FNfn10 allowed the introductionof four glycine residues while retaining the global fold. Insertionsin the AB and FG loops exhibited very small degrees of destabilization,comparable to or less than predicted entropic penalties dueto the elongation, suggesting the absence of stabilizing interactionsin these loops in wild-type FNfn10. Insertions in the BC, CDand DE loops, respectively, resulted in modest destabilization.In contrast, the EF loop elongation was highly destabilizing,consistent with previous studies showing the presence of stabilizinginteractions in this loop. These results suggest that all loops,except for the EF loop, can be used for engineering a bindingsite, thus demonstrating excellent properties of the monobodyscaffold.     

Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site

Organophosphorus hydrolase (OPH) is a metalloenzyme that can hydrolyze organophosphorus agents resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified three hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. We then experimentally assayed single and double mutants involving these residues for paraoxon binding affinity. The binding free energy calculations and the experimental kinetics of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced substrate binding affinity over WT OPH. Interestingly, our experimental results show that the substrate binding affinity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.     

A mutant D-amino acid aminotransferase with broad substrate specificity: construction by replacement of the interdomain loop Pro119- Arg120-Pro121 by Gly-Gly-Gly

D-amino acid aminotransferase (EC 2.6.1.21) catalyzes the interconversion of various D-amino acids and 2-oxo acids. Each homodimer subunit consists of two domains, which are connected by a single loop, Asn118-Pro119-Arg120-Pro121. The loop has no direct contact with the active site region or the cofactor, pyridoxal 5'- phosphate. We attempted to increase the conformational flexibility of this loop through a triple glycine substitution. The resultant mutant P119G-R120G-P121G has features clearly different from the wild-type enzyme under overall as well as half-reaction conditions. The pre- steady-state kinetic analyses of half reactions showed that the mutant enzyme has kmax values higher than the wild-type enzyme towards most D- amino acids examined. A concomitant decrease in substrate affinity (1/Kd), particularly for acidic amino acids, was also observed. A putative binding site for the distal carboxyl group of acidic amino acids in the wild-type enzyme was incidentally displaced by the loop mutation, indicating a functional linkage between the interdomain loop and the active site region. This study has exemplified the usefulness of engineering relatively distant loops as a means to modify substrate specificity of an enzyme.      

NaY分子筛中苯分子吸附和扩散行为的分子模拟

In the article the Grand Canonical Monte Carlo (GCMC), molecular dynamics (MD), and kinetic Monte Carlo (KMC) simulations with particular focus on ascertaining the loading dependence of benzene diffusion in the zeolite were performed. First, a realistic representation of the structure of the sorbate-sorbent system was obtained based on GCMC simulation. The simulation clearly shows the characteristics of the adsorption sites of the benzene-NaY system, from which two kinds of preferably adsorbing sites for benzene molecules, called SII and W sites, are identified. The structure thus obtained was then used as a basis for KMC and MD simulations. A compara-tive study by introducing and comparing two different mechanisms underlying jump diffusion in the zeolite of in-terest shows that the MS diffusivity values predicted by the KMC and MD methods are fairly close to each other, leading to the conclusion that for benzene diffusion in NaY, the SII→W→SII jumps of benzene molecules are dominated, while the W→W jumps do not exist in the process. These findings provide further support to our previous conclusion about the absence of the W→W jumps in the process of benzene diffusion in NaY. Finally, two relations for predicting the self- and MS diffusivities were derived and found to be in fair agreement with the KMC and MD simulations.     

Molecular Dynamics Studies on the Structural Characteristics for the Stability Prediction of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects the COVID-19 pandemic in the world. The spike protein of the various proteins encoded in SARS-CoV-2 binds to human ACE2, fuses, and enters human cells in the respiratory system. Spike protein, however, is highly variable, and many variants were identified continuously. In this study, Korean mutants for spike protein (D614G and D614A-C terminal domain, L455F and F456L-RBD, and Q787H-S2 domain) were investigated in patients. Because RBD in spike protein is related to direct interaction with ACE2, almost all researches were focused on the RBD region or ACE2-free whole domain region. The 3D structure for spike protein complexed with ACE2 was recently released. The stability analysis through RBD distance among each spike protein chain and the binding free energy calculation between spike protein and ACE2 were performed using MD simulation depending on mutant types in 1-, 2-, and 3-open-complex forms. D614G mutant of CT2 domain, showing to be the most prevalent in the global pandemic, showed higher stability in all open-complex forms than the wild type and other mutants. We hope this study will provide an insight into the importance of conformational fluctuation in the whole domain, although RBD is involved in the direct interaction with ACE2.     

Application of Molecular Dynamics Simulations in the Analysis of Cyclodextrin Complexes

Cyclodextrins (CDs) are highly respected for their ability to form inclusion complexes via host–guest noncovalent interactions and, thus, ensofance other molecular properties. Various molecular modeling methods have found their applications in the analysis of those complexes. However, as showed in this review, molecular dynamics (MD) simulations could provide the information unobtainable by any other means. It is therefore not surprising that published works on MD simulations used in this field have rapidly increased since the early 2010s. This review provides an overview of the successful applications of MD simulations in the studies on CD complexes. Information that is crucial for MD simulations, such as application of force fields, the length of the simulation, or solvent treatment method, are thoroughly discussed. Therefore, this work can serve as a guide to properly set up such calculations and analyze their results.     

The Disordered EZH2 Loop: Atomic Level Characterization by 1HN- and 1Hα-Detected NMR Approaches,Interaction with the Long Noncoding HOTAIR RNA

The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the ¹H^N-detected NMR approach presents many challenges under physiological conditions, our earlier developed, as well as improved, ¹H^α-detected experiments were used. As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407–Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2^T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR₁₄₀ (1–140 nt) highlight that the Thr367–Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein–RNA interaction as a “fuzzy” complex.     

Nanobody Paratope Ensembles in Solution Characterized by MD Simulations and NMR

Variable domains of camelid antibodies (so-called nanobodies or V_HH) are the smallest antibody fragments that retain complete functionality and therapeutic potential. Understanding of the nanobody-binding interface has become a pre-requisite for rational antibody design and engineering. The nanobody-binding interface consists of up to three hypervariable loops, known as the CDR loops. Here, we structurally and dynamically characterize the conformational diversity of an anti-GFP-binding nanobody by using molecular dynamics simulations in combination with experimentally derived data from nuclear magnetic resonance (NMR) spectroscopy. The NMR data contain both structural and dynamic information resolved at various timescales, which allows an assessment of the quality of protein MD simulations. Thus, in this study, we compared the ensembles for the anti-GFP-binding nanobody obtained from MD simulations with results from NMR. We find excellent agreement of the NOE-derived distance maps obtained from NMR and MD simulations and observe similar conformational spaces for the simulations with and without NOE time-averaged restraints. We also compare the measured and calculated order parameters and find generally good agreement for the motions observed in the ps–ns timescale, in particular for the CDR3 loop. Understanding of the CDR3 loop dynamics is especially critical for nanobodies, as this loop is typically critical for antigen recognition.     

Molecular dynamics studies on the thermostability of family 11 xylanases

Twelve members of the family 11 xylanases, including both mesophilic and thermophilic proteins, were studied using molecular dynamics (MD). Simulations of xylanases were carried out in an explicit water environment at four different temperatures, 300, 400, 500 and 600 K. A difference in thermotolerance between mesophilic and thermophilic xylanases became clear: thermophilic xylanases endured heat in higher simulation temperatures better than mesophilic ones. The unfolding pathways seemed to be similar for all simulations regardless of the protein. The unfolding initiates at the N-terminal region or alternatively from the alpha-helix region and proceeds to the 'finger region'. Unfolding of these regions led to denaturated structures within the 4.5 ns simulation at 600 K. The results are in agreement with experimental mutant studies. The results show clearly that the stability of the protein is not evenly distributed over the whole structure. The MD analysis suggests regions in the protein structure which are more unstable and thus potential targets for mutation experiments to improve thermostability.     

Decoding the Molecular Effects of Atovaquone Linked Resistant Mutations on Plasmodium falciparum Cytb-ISP Complex in the Phospholipid Bilayer Membrane

Atovaquone (ATQ) is a drug used to prevent and treat malaria that functions by targeting the Plasmodium falciparum cytochrome b (PfCytb) protein. PfCytb catalyzes the transmembrane electron transfer (ET) pathway which maintains the mitochondrial membrane potential. The ubiquinol substrate binding site of the protein has heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors that act as redox centers to aid in ET. Recent studies investigating ATQ resistance mechanisms have shown that point mutations of PfCytb confer resistance. Thus, understanding the resistance mechanisms at the molecular level via computational approaches incorporating phospholipid bilayer would help in the design of new efficacious drugs that are also capable of bypassing parasite resistance. With this knowledge gap, this article seeks to explore the effect of three drug resistant mutations Y268C, Y268N and Y268S on the PfCytb structure and function in the presence and absence of ATQ. To draw reliable conclusions, 350 ns all-atom membrane (POPC:POPE phospholipid bilayer) molecular dynamics (MD) simulations with derived metal parameters for the holo and ATQ-bound -proteins were performed. Thereafter, simulation outputs were analyzed using dynamic residue network (DRN) analysis. Across the triplicate MD runs, hydrophobic interactions, reported to be crucial in protein function were assessed. In both, the presence and absence of ATQ and a loss of key active site residue interactions were observed as a result of mutations. These active site residues included: Met 133, Trp136, Val140, Thr142, Ile258, Val259, Pro260 and Phe264. These changes to residue interactions are likely to destabilize the overall intra-protein residue communication network where the proteins’ function could be implicated. Protein dynamics of the ATQ-bound mutant complexes showed that they assumed a different pose to the wild-type, resulting in diminished residue interactions in the mutant proteins. In summary, this study presents insights on the possible effect of the mutations on ATQ drug activity causing resistance and describes accurate MD simulations in the presence of the lipid bilayer prior to conducting inhibitory drug discovery for the PfCytb-iron sulphur protein (Cytb-ISP) complex.     

Magnetite‐Binding Flagellar Filaments Displaying the MamI Loop Motif

This work aimed at developing a novel method for fabricating 1 D magnetite nanostructures with the help of mutated flagellar filaments. We constructed four different flagellin mutants displaying magnetite‐binding motifs: two contained fragments of magnetosome‐associated proteins from magnetotactic bacteria (MamI and Mms6), and synthetic sequences were used for the other two. A magnetic selection method identified the MamI mutant as having the highest binding affinity to magnetite. Filaments built from MamI loop‐containing flagellin subunits were used as templates to form chains of magnetite nanoparticles along the filament by capturing them from suspension. Our study represents a proof‐of‐concept that flagellar filaments can be engineered to facilitate formation of 1 D magnetite nanostructures under ambient conditions. In addition, it proves the interaction between MamI and magnetite, with implications for the role of this protein in magnetotactic bacteria.     

Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin

The loop exchange mutant chymosm 155–164 rhizopuspepsinwas expressed in Trichoderma reesei and exported into the mediumto yield a correctly folded and active product. The biochemicalcharacterization and crystal structure determination at 2.5Å resolution confirm that the mutant enzyme adopts a nativefold. However, the conformation of the mutated loop is unlikethat in native rhizopuspepsin and involves the chelation ofa water molecule in the loop. Kinetic analysis using two syntheticpeptide substrates (six and 15 residues long) and the naturalsubstrate, milk, revealed a reduction in the activity of themutant enzyme with respect to the native when acting on boththe long peptide substrate and milk. This may be a consequenceof the different charge distribution of the mutated loop, itsincreased size and/or its different conformation.     

Stepwise transplantation of an active site loop between heat-labile enterotoxins LT-II and LT-I and characterization of the obtained hybrid toxins

Members of the cholera toxin family, including Escherichia coli heat- labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from NAD to a specific arginine of the target protein. The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity. The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft. Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I. Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I. Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT- II. Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site. The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I. Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I. The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I. Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity. The most striking result of this study is that the active site loop of LT- I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well as 'hybrid toxin'.      

In silico mutations and molecular dynamics studies on a winged bean chymotrypsin inhibitor protein

Winged bean chymotrypsin inhibitor (WCI) has an intruding residueAsn14 that plays a crucial role in stabilizing the reactivesite loop conformation. This residue is found to be conservedin the Kunitz (STI) family of serine protease inhibitors. Tounderstand the contribution of this scaffolding residue on thestability of the reactive site loop, it was mutated in silicoto Gly, Ala, Ser, Thr, Leu and Val and molecular dynamics (MD)simulations were carried out on the mutants. The results ofMD simulations reveal the conformational variability and rangeof motions possible for the reactive site loop of differentmutants. The N-terminus side of the scissile bond, which isclose to a ß-barrel, is conformationally less variable,while the C-terminus side, which is relatively far from anysuch secondary structural element, is more variable and needsstability through hydrogen-bonding interactions. The simulatedstructures of WCI and the mutants were docked in the peptide-bindinggroove of the cognate enzyme chymotrypsin and the ability toform standard hydrogen-bonding interactions at P3, P1 and P2'residues were compared. The results of the MD simulations coupledwith docking studies indicate that hydrophobic residues likeLeu and Val at the 14th position are disruptive for the integrityof the reactive site loop, whereas a residue like Thr, whichcan stabilize the C-terminus side of the scissile bond, canbe predicted at this position. However, the size and chargeof the Asn residue made it most suitable for the best maintenanceof the integrity of the reactive site loop, explaining its conservednature in the family. Received October 17, 2002; revised June 6, 2003; accepted June 19, 2003.