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Modeling the thermodynamics of a transition metal (TM) ion assembly be it in proteins or in coordination complexes affords us a better understanding of the assembly and function of metalloclusters in diverse application areas including metal organic framework design, TM-based catalyst design, the trafficking of TM ions in biological systems, and drug design in metalloprotein platforms. While the structural details of TM ions bound to metalloproteins are generally well understood via experimental and computational approaches, accurate studies describing the thermodynamics of TM ion binding are rare. Herein, we demonstrate that we can obtain accurate structural and absolute binding free energies of Co 2+ and Ni 2+ to the enzyme glyoxalase I using an optimized 12-6-4 (m12-6-4) potential. Critically, this model simultaneously reproduces the solvation free energy of the individual TM ions and reproduces the thermodynamics of TM ion-ligand coordination as well as the thermodynamics of TM ion binding to a protein active site unlike extant models. We find the incorporation of the thermodynamics associated with protonation state changes for the TM ion (un)binding to be crucial. The high accuracy of m12-6-4 potential in this study presents an accurate route to explore more complicated processes associated with TM cluster assembly and TM ion transport.

Despite the rich history of experimental studies focusing on the thermochemistry and kinetics associated with the chelate effect, molecular-level computational studies on the chelate ring opening/ring closure are scarce. The challenge lies in an accurate description of both the metal ion and its aqueous environment. Herein, we demonstrate that an optimized 12-6-4 Lennard-Jones (LJ) model can capture the thermodynamics and provide detailed structural and mechanistic insights into the formation of ethylenediamine (en) complexes with metal ions. The water molecules in the first solvation shell of the metal ion are found to facilitate the chelate ring formation. The optimized parameters further simulate the formation of bis and tris(en) complexes representing the wide applicability of the model to simulate coordination chemistry and self-assembly processes.

Obtaining a detailed description of how active site flap motion affects substrate or ligand binding will advance structure-based drug design (SBDD) efforts on systems including the kinases, HSP90, HIV protease, ureases, etc. Through this understanding, we will be able to design better inhibitors and better proteins that have desired functions. Herein we address this issue by generating the relevant configurational states of a protein flap on the molecular energy landscape using an approach we call MT Flex-b and then following this with a procedure to estimate the free energy associated with the motion of the flap region. To illustrate our overall workflow, we explored the free energy changes in the streptavidin/biotin system upon introducing conformational flexibility in loop 3-4 in the biotin unbound ( apo) and bound ( holo) state. The free energy surfaces were created using the Movable Type free energy method, and for further validation, we compared them to potential of mean force (PMF) generated free energy surfaces using MD simulations employing the FF99SBILDN and FF14SB force fields. We also estimated the free energy thermodynamic cycle using an ensemble of closed-like and open-like end states for the ligand unbound and bound states and estimated the binding free energy to be approximately -16.2 kcal/mol (experimental -18.3 kcal/mol). The good agreement between MT Flex-b in combination with the MT method with experiment and MD simulations supports the effectiveness of our strategy in obtaining unique insights into the motions in proteins that can then be used in a range of biological and biomedical applications.

How to fine-tune the binding free energy of a small-molecule to a receptor site by altering the amino acid residue composition is a key question in protein engineering. Indeed, the ultimate solution to this problem, to chemical accuracy (±1 kcal/mol), will result in profound and wide-ranging applications in protein design. Numerous tools have been developed to address this question using knowledge-based models to more computationally intensive molecular dynamics simulations-based free energy calculations, but while some success has been achieved there remains room for improvement in terms of overall accuracy and in the speed of the methodology. Here we report a fast, knowledge-based movable-type (MT)-based approach to estimate the absolute and relative free energy of binding as influenced by mutations in a small-molecule binding site in a protein. We retrospectively validate our approach using mutagenesis data for retinoic acid binding to the Cellular Retinoic Acid Binding Protein II (CRABPII) system and then make prospective predictions that are borne out experimentally. The overall performance of our approach is supported by its success in identifying mutants that show high or even sub-nano-molar binding affinities of retinoic acid to the CRABPII system.

A metal-mediated interprotomer hydrogen bond has been implicated in the allosteric mechanism of DNA operator binding in several metal-sensing proteins. Using computational methods, we investigate the energetics of such zinc-mediated interactions in members of the ArsR/SmtB family of proteins (CzrA, SmtB, CadC, and NmtR) and the MarR family zinc-uptake repressor AdcR, which feature similar interactions, but in sites that differ widely in their allosteric responsiveness. We provide novel structural insight into previously uncharacterized allosteric forms of these proteins using computational methodologies. We find this metal-mediated interaction to be significantly stronger (∼8 kcal/mol) at functional allosteric metal binding sites compared to a nonresponsive site (CadC) and the apo-proteins. Simulations of the apo-proteins further reveal that the high interaction energy works to overcome the considerable disorder at these hydrogen-bonding sites and functions as a "switch" to lock in a weak DNA-binding conformation once metal is bound. These findings suggest a conserved functional role of metal-mediated second coordination shell hydrogen bonds at allosterically responsive sites in zinc-sensing transcription regulators.

Substrate ingress and product egress from the active site of urease is tightly controlled by an active-site flap. Molecular dynamics simulations of urease have revealed a previously unobserved wide-open flap state that, unlike the well-characterized closed and open states, allows ready access to the metal cluster in the active site. This state is easily reached from the open state via low free energy barriers. Additionally, we have found that even when the flap is closed, a region of the binding pocket is solvent-exposed, leading to the hypothesis that it may act as a substrate/product reservoir. The newly identified wide-open state offers further opportunities for small-molecule drug discovery by defining a more extensive active-site pocket than has been previously described.

The zinc sensing transcriptional repressor Staphylococcus aureus CzrA represents an excellent model system to understand how metal sensor proteins maintain cellular metal homeostasis. Zn(II) binding induces a quaternary structural switch from a "closed" conformation to a more "open" conformation, reducing the DNA binding affinity by 4 orders of magnitude. In this study, we use classical molecular dynamics and quantum mechanical/molecular mechanical molecular dynamics simulations to investigate the molecular basis for the large conformational motions and allosteric coupling free energy (~6 kcal/mol) associated with Zn(II) binding. Our simulations successfully capture the closed to open allosteric switching in DNA bound CzrA on Zn(II) binding. They reveal that zinc binding quenches global conformational sampling by CzrA, whereas DNA binding enhances the mobility of residues in the allosteric metal binding sites. These findings are in close agreement with experiments. We also identify networks of residues involved in correlated and anticorrelated motions that connect the metal binding and DNA binding sites. Our analysis of the essential dynamics shows metal ion binding to be the primary driving force for the quaternary structural change in CzrA. We also show that Zn(II) binding limits the conformational space sampled by CzrA and causes the electrostatic surface potential at the DNA binding interface to become less favorable toward DNA binding. Finally, our simulations provide strong support for a proposed hydrogen-bonding pathway that physically connects the metal binding residue, His97, to the DNA binding interface through the αR helix that is present only in the Zn(II)-bound state. Overall, our simulations provide molecular-level insights into the mechanism of allosteric regulation by CzrA and demonstrate the importance of protein motion in its biological activity.

Protein farnesyltransferase (FTase) catalyzes farnesylation of a variety of peptide substrates. (3)H α-secondary kinetic isotope effect (α-SKIE) measurements of two peptide substrates, CVIM and CVLS, are significantly different and have been proposed to reflect a rate-limiting S(N)2-like transition state with dissociative characteristics for CVIM, while, due to the absence of an isotope effect, CVLS was proposed to have a rate-limiting peptide conformational change. Potential of mean force quantum mechanical/molecular mechanical studies coupled with umbrella sampling techniques were performed to further probe this mechanistic dichotomy. We observe the experimentally proposed transition state (TS) for CVIM but find that CVLS has a symmetric S(N)2 TS, which is also consistent with the absence of a (3)H α-SKIE. These calculations demonstrate facile substrate-dependent alterations in the transition state structure catalyzed by FTase., (© 2011 American Chemical Society)

The periplasmic Cu(+)/Ag(+) chaperone CusF features a novel cation-π interaction between a Cu(+)/Ag(+) ion and Trp44 at the metal binding site. The nature and strength of the Cu(+)/Ag(+)-Trp44 interactions were investigated using computational methodologies. Quantum-mechanical (QM) calculations showed that the Cu(+) and Ag(+) interactions with Trp44 are of similar strength (~14 kcal/mol) and bond order. Quantum-mechanical/molecular-mechanical (QM/MM) calculations showed that Cu(+) binds in a distorted tetrahedral coordination environment in the Trp44Met mutant, which lacks the cation-π interaction. Molecular dynamics (MD) simulations of CusF in the apo and Cu(+)-bound states emphasized the importance of the Cu(+)-Trp44 interaction in protecting Cu(+) from water oxidation. The protein structure does not change over the time scale of hundreds of nanoseconds in the metal-bound state. The metal recognition site exhibits small motions in the apo state but remains largely preorganized toward metal binding. Trp44 remains oriented to form the cation-π interaction in the apo state and faces an energetic penalty to move away from the metal ion. Cu(+) binding quenches the protein's internal motions in regions linked to binding CusB, suggesting that protein motions play an essential role in Cu(+) transfer to CusB., (© 2011 American Chemical Society)

Pairwise decomposition of the interaction energy between molecules is shown to be a powerful tool that can increase our understanding of macromolecular recognition processes. Herein we calculate the pairwise decomposition of the interaction energy between the protein human carbonic anhydrase II (HCAII) and the fluorine-substituted ligand N-(4-sulfamylbenzoyl)benzylamine (SBB) using semiempirical quantum mechanics based methods. We dissect the interaction between the ligand and the protein by dividing the ligand and the protein into subsystems to understand the structure-activity relationships as a result of fluorine substitution. In particular, the off-diagonal elements of the Fock matrix that is composed of the interaction between the ionic core and the valence electrons and the exchange energy between the subsystems or atoms of interest is examined in detail. Our analysis reveals that the fluorine-substituted benzylamine group of SBB does not directly affect the binding energy. Rather, we find that the strength of the interaction between Thr199 of HCAII and the sulfamylbenzoyl group of SBB affects the binding affinity between the protein and the ligand. These observations underline the importance of the sulfonamide group in binding affinity as shown by previous experiments (Maren, T. H.; Wiley: C. E. J. Med. Chem. 1968, 11, 228-232). Moreover, our calculations qualitatively agree with the structural aspects of these protein-ligand complexes as determined by X-ray crystallography.

We have calculated proton chemical shift perturbations (CSPs) of retinol in the cellular retinol-binding protein (CRBP) through the use of a recently developed computational approach (Wang et al. J. Chem. Phys. 2004, 120, 11392-11400). Excellent agreement with experimental values was obtained for the X-ray structure, whereas the lack of a key hydrogen bond and the distorted isoprene tail of retinol for some NMR models lead to large CSP RMSDs. Therefore, a comparison of computed CSPs of retinol with experiment offers a convenient way to validate the structure of retinol and its orientation in the binding site for the NMR structures.

Based on hybrid QM/MM molecular dynamics simulation and density functional theoretical (DFT) calculations, we investigate the mechanistic and energetic features of the catalytic action of dizinc metallo-beta-lactamase CcrA from Bacteroides fragilis. The 200 ps QM/MM simulation of the CcrA enzyme in complex with nitrocefin shows that the substrate beta-lactam moiety is directed toward the active site dizinc center through the interactions of aminocarbonyl and carboxylate groups with the two active site zinc ions and the two conserved residues, Lys167 and Asn176. From the determination of the potential energy profile of a relevant enzymatic reaction model, it is found that the nucleophilic displacement reaction step proceeds with a low-barrier height, leading to the formation of an energetically favored reaction intermediate. The results also show that the high catalytic activity of the CcrA enzyme stems from a simultaneous operation of three catalytic components: activation of the bridging hydroxide nucleophile by zinc-coordinated Asp86; polarization of the substrate aminocarbonyl group by the first zinc ion; stabilization of the negative charge developed on the departing amide nitrogen by the second zinc ion. Consistent with the previous experimental finding that the proton-transfer reaction step is rate-limiting, the activation energy of the second step is found to be 1.6 kcal/mol higher than that of the first step. Finally, through an examination of the structural and energetic features of binding of a thiazolidinecarboxylic acid inhibitor to the active site dizinc center, a two-step inhibition mechanism involving a protonation-induced ligand exchange reaction is proposed for the inhibitory action of a tight-binding inhibitor possessing a thiol group.

We present a high-level quantum chemical study of possible reaction mechanisms associated with the catalytic decomposition of urea by a bioinorganic mimetic of the dinickel active site of urease. We chose the phthalazine-dinickel complexes of Lippard and co-workers, because these mimetics have been shown to hydrolytically degrade urea. High-level quantum chemical methodologies were utilized to identify stable intermediates and transition-state structures along several possible reaction pathways. The computed results were then used to further analyze what may occur in the active site of urease. Valuable information on the latter has been extracted from experimental data, computational approaches, and unpublished molecular dynamics simulations. On the basis of these comparative studies, we propose that both the elimination and hydrolytic pathways may compete for urea decomposition in the active site of urease.

Recently, we have developed a fast approach to calculate NMR chemical shifts using the divide and conquer method at the semiempirical level. To demonstrate the utility of this approach for characterizing protein-ligand interactions, we used the deviation of calculated chemical shift perturbations from experiment to determine the orientation of a ligand (GPI-1046) in the binding pocket of the FK506 binding protein (FKBP12). Moreover, we were able to select the native state of the ligand from a collection of decoy poses. A key hydrogen bond between O1 and HN in Ile56 was also identified. Our results suggest that ligand-induced chemical shift perturbations can be used to refine protein/ligand structures.

We present the results of a computational study of the solution phase decomposition of urea, which provides insight into probable reaction pathways for the urease-catalyzed reaction. Calculations, which were used to derive thermodynamic parameters that were further used for a kinetic analysis, have been done at the solvent-corrected MP2/6-311++G** level. Both elimination and hydrolytic pathways have been considered. Elimination is favored for the solution phase reaction, which proceeds by H-bond coordination of a water molecule to the amine nitrogen atoms. The coordination of one water molecule greatly facilitates the reaction by allowing it to proceed through a cyclic six-member transition state. Aspects of the water-urea H-bond interactions have also provided insights into critical aspects of the hydrogen bond pattern in the urease active site. On the basis of a kinetic analysis, we have estimated the proficiency of urease and have predicted that it is the most proficient enzyme identified to date.

In this communication, we report the development of a novel quantum mechanics-based scoring function to predict free energy of ligand binding in the zinc metalloenzymes carbonic anhydrase (CA) and carboxypeptidase A (CPA). In particular, the AM1 method is used in conjunction with solvation modeling to predict the relative binding affinities of 18 CA and 5 CPA inhibitors. The effect of metal-ligand charge transfer is also discussed and shown to be different in CPA and CA, providing a further challenge to computing metalloenzyme binding affinities.

Herein, we present results from a computational study of dinickel complexes that are relevant to the catalytic hydrolysis of urea exerted by the urease enzymes. The B3LYP density functional is used to characterize the equilibrium geometry, electronic and magnetic properties, and energies for a series of realistic complexes modeling the active site of ureases. The analysis of the theoretical results gives new insight into the structure, substrate binding, and catalytic mechanism. The water bridge between the two Ni(II) ions observed in the crystallographic structures of the ureases was assigned to a hydroxide bridge in agreement with the observed small antiferromagnetic coupling. Both monodentate and bidentate urea-bound complexes, in which urea had favorable orientations for catalysis, were characterized. Finally, two reaction mechanisms were investigated starting from the monodentate and bidentate urea-bound complexes, respectively. Both a Ni1...Ni2 bridging hydroxide and a Ni2-bound water molecule play crucial roles in the two mechanisms.

To further understand Delta(5)-3-ketosteroid isomerase (KSI) catalysis, we carried out molecular dynamics (MD) simulations of the KSI dimer ligated with a substrate and reaction intermediate analogue and high level ab initio calculations on relevant enzymatic reaction models. Simulation of the enzyme-substrate complex dimer systems showed asymmetric dynamics between the two monomers, in which the hydrogen bond pattern between the substrate and active site residues in the first and the second subunits supported the cooperative hydrogen bond (CH) and the catalytic diad (CD) mechanisms, respectively. On the other hand, only the CH mechanism was supported in the MD simulation of the enzyme-intermediate complex dimer. From MP2/6-31+G**//RHF/6-31G** calculations, we found the kinetic barriers for the two reaction mechanisms were similar. The CH route afforded a greater stabilization to the enolate intermediate than did the CD counterpart. Thus, the present computational studies indicate that the CH mechanism would be favored over the CD one in the catalytic action of KSI. However, the latter could not be ruled out conclusively because of the explicit appearance of a CD configuration in the MD trajectories of the enzyme-substrate complex and because of the similar intrinsic activation barrier for the CH and CD mechanisms. The appearance of configurations that favor the CD pathway is rationalized in terms of a model in which the KSI-substrate complex does not have a strong preference for one hydrogen bonding pattern over another, while the KSI-intermediate complex favors a cooperative hydrogen bond pattern in order to stabilize the reaction intermediate. This hypothesis is supported by the ab initio calculations which indicate that the CH intermediate is more stable than the CD one by approximately 6.3 kcal/mol.

Herein, we present results from molecular dynamics MD simulations ( approximately 1 ns) of the TEM-1 beta-lactamase in aqueous solution. Both the free form of the enzyme and its complex with benzylpenicillin were studied. During the simulation of the free enzyme, the conformation of the Omega loop and the interresidue contacts defining the complex H-bond network in the active site were quite stable. Most interestingly, the water molecule connecting Glu166 and Ser70 does not exchange with bulk solvent, emphasizing its structural and catalytic relevance. In the presence of the substrate, Ser130, Ser235, and Arg244 directly interact with the beta-lactam carboxylate via H-bonds, whereas the Lys234 ammonium group has only an electrostatic influence. These interactions together with other specific contacts result in a very short distance ( approximately 3 A) between the attacking hydroxyl group of Ser70 and the beta-lactam ring carbonyl group, which is a favorable orientation for nucleophilic attack. Our simulations also gave insight into the possible pathways for proton abstraction from the Ser70 hydroxyl group. We propose that either the Glu166 carboxylate-Wat1 or the substrate carboxylate-Ser130 moieties could abstract a proton from the nucleophilic Ser70.

Herein, we present results from MD simulations of the Michaelis complex formed between the B. cereus zinc-beta-lactamase enzyme and benzylpenicillin. The structural and dynamical effects induced by substrate-binding, the specific role of the conserved residues, and the near attack conformers of the Michaelis complex are discussed. Quantum chemical methods (HF/6-31G* and B3LYP/6-31G*) are also applied to study the hydrolysis reaction of N-methylazetidinone catalyzed by a monozinc system consisting of the side chains of the histidine residues (His86, His88, and His149) complexed with Zn-OH and the side chains of Asp90 and His210. From this model system, we built molecular-mechanics representations of the prereactive complex and transition state configurations docked into the active site. Linear-scaling semiempirical calculations coupled with a continuum solvent model were then performed on these static models. We propose that the experimental rate data for the B. cereus enzyme is compatible with a one-step mechanism for the hydrolysis of beta-lactam substrates in which His210 acts as a proton donor.

Herein we report the results of a HF/6-31+G** and B3LYP/6-31+G** computational investigation of the title reaction for the peptide bond synthesis catalyzed by a single adenine. This system constitutes a reference reaction to study the basic chemical events that have been proposed to occur at the peptidyl transferase active site of ribosomes on the basis of structural determinations (Science 2000, 239, 920--931). Thus, the analysis of the geometry, charge distribution, and energetics of the critical structures involved in this title reaction yields insight into the catalytic mode of action of RNA molecules. Our computational results give further support to the hypotheses that the activated nucleotide A2451 in the ribosome acts as a base catalyst and that this role is similar to that of the His residue in the catalytic triad of serine proteases.

Herein, we present results of a computational study on benzylpenicillin attachment to Lys199 of human serum albumin via an aminolysis reaction. The internal geometry of the reactive part of the system was taken from previous work at the B3LYP/6-31+G* level on the water-assisted aminolysis reaction of a penicillin model compound (Díaz, N.; Suárez, D.; Sordo, T. L. J. Am. Chem. Soc. 2000, 122, 6710--6719). The protein environment around Lys199, the 6-acylamino side chain, and the 2-methyl groups of benzylpenicillin were relaxed by carrying out geometry optimizations with a hybrid QM/MM method (PM3/AMBER). Two different mechanistic routes were explored: a one-step water-assisted process and a carboxylate and water-assisted route in which the beta-lactam carboxylate and the ancillary water molecule mediate the proton transfer from the epsilon-amino group of Lys199 to the beta-lactam leaving N atom. The corresponding energy profiles in the protein combine the B3LYP/6-31+G* and PM3 energies of the reactive subsystem (benzylpenicillin + Lys199 side chain + the ancillary water molecule) and semiempirical PM3 energies of the entire system evaluated with a "divide and conquer" linear-scaling method. It is observed that penicillin haptenation to HSA can proceed through the water-assisted concerted mechanism which is calculated to have a high energy barrier of approximately 38 kcal/mol, in agreement with the experimentally observed slow reaction kinetics.

The catalytic mechanism of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase) is evaluated with pH dependent kinetics, site-directed mutagenesis, and quantum chemical calculations. The chemistry step, represented by the burst rates, was not pH-dependent, which is consistent with our proposed mechanism that the 4-carboxamide of AICAR assists proton shuttling. Quantum chemical calculations on a model system of 5-amino-4-carboxamide imidazole (AICA) and formamide using the B3LYP/6-31G level of theory confirmed that the 4-carboxamide participated in the proton-shuttling mechanism. The result also indicated that the amide-assisted mechanism is concerted such that the proton transfers from the 5-amino group to the formamide are simultaneous with nucleophilic attack by the 5-amino group. Because the process does not lead to a kinetically stable intermediate, the intramolecular proton transfer from the 5-amino group through the 4-carboxamide to the formamide proceeds in the same transition state. Interestingly, the calculations predicted that protonation of the N3 of the imidazole of AICA would reduce the energy barrier significantly. However, the pK(a) of the imidazole of AICAR was determined to be 3.23 +/- 0.01 by NMR titration, and AICAR is likely to bind to the enzyme with its imidazole in the free base form. An alternative pathway was suggested by modeling Lys266 to have a hydrogen-bonding interaction with the N3 of the imidazole of AICAR. Lys266 has been implicated in catalysis based on mutagenesis studies and the recent X-ray structure of AICAR Tfase. The quantum chemical calculations on a model system that contains AICA complexed with CH3NH3+ as a mimic of the Lys residue confirmed that such an interaction lowered the activation energy of the reaction and likewise implicated the 4-carboxamide. To experimentally verify this hypothesis, we prepared the K266R mutant and found that its kcat is reduced by 150-fold from that of the wild type without changes in substrate and cofactor Km values. The kcat-pH profile indicated virtually no pH-dependence in the pH range 6-10.5. The results suggest that the ammonium moiety of Lys or Arg is important in catalysis, most likely acting as a general acid catalyst with a pK(a) value greater than 10.5. The H267A mutant was also prepared since His267 has been found in the active site and implicated in catalysis. The mutant enzyme showed no detectable activity while retaining its binding affinity for substrate, indicating that it plays a critical role in catalysis. We propose that His267 interacts with Lys266 to aid in the precise positioning of the general acid catalyst to the N3 of the imidazole of AICAR.

Herein, we report molecular dynamics simulations of the mononuclear form of the Bacillus cereuszinc-beta-lactamase. We studied two different configurations which differ in the presence of a zinc-bound hydroxide or a zinc-bound water and in the protonation state of the essential His210 residue. Contacts of the catalytically important residues (Asp90, His210, Cys168, etc.) with the zinc center are characterized by the MD analyses. The nature of the Zn-OH(2) --> His210 proton transfer pathway connecting the two configurations was studied by means of QM calculations on cluster models while the relative stability of the two configurations was estimated from QM/MM calculations in the enzyme. From these results, a theoretical model for the kinetically active form of the B. cereus metalloenzyme is proposed. Some mechanistic implications and the influence of mutating the Cys168 residue are also discussed.