We have generated a highly selective cathepsin B probe and several less specific reagents for the study of cathepsin biology. The reagents have several advantages over commonly used fluorogenic substrates, allowing inhibitor targets to be identified in a pool of total cellular enzymes. We have used the probes to show that cathepsin activity is regulated in tumor tissues and during differentiation of placental-derived cytotrophoblasts to invasive cells required for establishing blood circulation in a developing embryo.

In this paper we describe the first all-atom aqueous-phase MD simulations of human carbonic anhydrase II in three protonation states relevant to the rate-limiting intramolecular proton-transfer step. In particular, we have examined the zinc−water form of the enzyme (CHOH), the zinc−hydroxide form of the enzyme with a doubly protonated His-64 (COHH, the putative intramolecular proton-transfer proton-accepting residue), and the native zinc−hydroxide form (COH) of the enzyme (i.e., with an unprotonated His-64). From these MD simulations (up to ∼1 ns in length) we have studied in detail the dynamics of these three systems. Overall the dynamics of the three systems do not vary significantly (e.g., the active site region is rigid, the number of long-lived hydrogen bonds is constant, etc.) with the exception of COHH. In this case the residues that line the entrance to the active site cavity (near the location of His-64) undergo significantly higher fluctuations than in the CHOH and COH cases. It is postulated that this facilitates solvent and buffer exchange around His-64, thereby facilitating the intermolecular proton-transfer step. We also find that the motion of His-64 is limited in all three cases to occupying the “in” orientation (∼7 Å from the zinc ion, while the so-called “out” conformer is further away), which suggests that fluctuations of this residue between the in and out conformers have a limited influence on the intramolecular proton transfer. However, due to the limited time scales of our simulations, this needs to be examined in more detail. Importantly, though, we find that His-64 acts as a “gate-keeper” between the inner active site region (characterized by localized water molecules) and the outer (bulk) region, which is characterized by relatively freely diffusing water molecules. This function of His-64 has not been realized previously. In the inner active site we have identified relatively long-lived water bridges between the zinc-bound water or hydroxide and the imidazole or imidazolium side chain of His-64. The lengths of these bridges vary between two and six water molecules, and the preferred bridge depends on the protonation of the active site. We estimate that the probability of water bridge formation is low (at most ∼1.5 kcal/mol) and that water bridge formation is not the rate-limiting step in the proton-transfer process (transfer from zinc-bound water to an active site water is rate-limiting).

Classifying proteins into functionally distinct families based only on primary sequence information remains a difficult task. We describe here a method to generate a large data set of small molecule affinity fingerprints for a group of closely related enzymes, the papain family of cysteine proteases. Binding data was generated for a library of inhibitors based on the ability of each compound to block active-site labeling of the target proteases by a covalent activity based probe (ABP). Clustering algorithms were used to automatically classify a reference group of proteases into subfamilies based on their small molecule affinity fingerprints. This approach was also used to identify cysteine protease targets modified by the ABP in complex proteomes by direct comparison of target affinity fingerprints with those of the reference library of proteases. Finally, experimental data were used to guide the development of a computational method that predicts small molecule inhibitors based on reported crystal structures. This method could ultimately be used with large enzyme families to aid in the design of selective inhibitors of targets based on limited structural/function information.

Matrix metalloproteinases (MMPs) represent a potentially important class of therapeutic targets for the treatment of diseases such as cancer. Selective inhibition of MMPs will be required given the high sequence identity across the family and the discovery that individual MMPs also regulate the natural angiogenesis inhibitor angiostatin. In this study, we have used computational methods to model the selectivity for six thiadiazole urea inhibitors with stromelysin-1 and gelatinase-A, two homologous MMPs that have been implicated in breast cancer. From continuum Generalized Born molecular dynamics (GB-MD) and MM-GBSA analysis, we estimated ligand free energies of binding using 200 snapshots obtained from a short 40 ps simulation of the relevant protein-ligand complex. The MM-GBSA free energies, computed from the continuum GB-MD trajectories, show strong correlation with the experimental affinities (r(2) = 0.74); prior studies have employed explicit water MD simulations. Including estimates for changes in solute entropy in the binding calculations slightly diminishes the overall correlation with experiment (r2 = 0.71). Notably, in every case, the simulation results correctly predict that a given ligand will bind selectively to stromelysin-1 over gelatinase-A which is gratifying given the high degree of structural homology between the two proteins. The increased selectivity for stromelysin-1 appears to be driven by (1) increased favorable van der Waals interactions, (2) increased favorable Coulombic interactions, and (3) decreased unfavorable total electrostatic energies (Coulombic plus desolvation).

We have investigated the serine protease γ-chymotrypsin (γ-CT) in three different solvation environments using molecular dynamics simulations. These solvation environments include the following:  (1) γ-CT taken from the crystal structure of Yennawar et al. (Biochemistry 1994, 33, 7326−7336) with seven surface bound hexane molecules and 50 bound “essential” water molecules all immersed in 1109 hexane molecules (simulation labeled CT); (2) γ-CT taken from Yennawar et al. and solvated with 50 “essential” water molecules and immersed in 1107 hexane molecules (simulation labeled CTWAT); and (3) γ-CT taken from Yennawar et al. and solvated with a monolayer of 444 water molecules and immersed in 931 hexane molecules (simulation labeled CTMONO). From these trajectories we found that the placement of bound water molecules and the amount of hydration of the protein in the simulated structure had an effect on the protein flexibility as indicated by changes in the RMS deviation. The radius of gyration value was similar for the three systems, indicating no significant unfolding or denaturation. Hydrophobic residues were found to have increased solvent accessible surface area (SASA), while hydrophilic residues experienced a decrease in SASA in the CT and CTWAT simulations. No hexane diffusion into the protein interior was found, with the exception of one bound hexane site in the CT simulation. The secondary structure analysis of the active site indicates that the active site structure was retained in all three simulations. We also found that intramolecular forces (i.e., hydrogen bonding) that stabilize proteins are stronger in the CT and CTWAT systems, as shown by the increase in the number of stable hydrogen bonds found. Net ion pair interactions and reduced ratio of surface area to volume of the protein also contributed to the stability of the protein in anhydrous organic media.

In this paper we report molecular dynamics (MD) and free energy perturbation (FEP) studies carried out on enzyme-inhibitor (two hydroxamates that only differ by a carbon-carbon double bond) complexes of human fibroblast collagenase to obtain insights into the structural and energetic preferences of these inhibitors. We have developed a bonded model for the catalytic and structural zinc centers (Hoops, S. C.; et al. J. Am. Chem. Soc. 1991, 113, 8262-8270) where the electrostatic representation for this model was derived using a novel quantum-mechanical/molecular-mechanical (QM/MM) minimization procedure followed by electrostatic potential fitting. The resulting bonded model for the zinc ions was then used to generate MD trajectories for structural analysis and FEP studies. This model has satisfactorily reproduced the structural features of the active site, and furthermore, the FEP simulations gave relative free energies of binding in good agreement with experimental results. MD simulations in conjunction with the FEP are able to provide a structural explanation regarding why one hydroxamate inhibitor is favored over the other, and we are also able to make predictions about changes in the inhibitor that would enhance protein-inhibitor interactions.

Herein we examine the origin of enantioselectivity in the serine protease subtilisin in DMF through the use of molecular dynamics (MD) and free energy perturbation (FEP) simulations. In particular, we are interested in the resolution of a racemic mixture of sec-phenethyl alcohol by a transesterification reaction with the acylating agent vinyl acetate, catalyzed by subtilisin in anhydrous dimethylformamide (DMF). To study the enantioselectivity in this case, we examined the tetrahedral intermediate as a model of the enzyme transition state (as has been done in the past). A critical aspect of this study was the determination of the charge distribution of the two (R and S) tetrahedral intermediates through the use of a combined quantum mechanical/molecular mechanical electrostatic potential fitting methodology. In designing the active site charge model, we found that the R and S tetrahedral intermediates have significantly different charge distributions due to the presence of the stereodifferentiating environment presented by the enzyme. In contrast the charge distribution obtained for models of the tetrahedral intermediate in the gas phase have similar charge distributions. From MD simulations we find that both steric and electrostatic complimentarity plays a role in the enantioselectivity of this enzyme-catalyzed reaction. Through the use of FEP simulations we obtained a free energy difference that is in good accord with experiment, which quantitatively supports the accuracy of our model and suggests that all-atom molecular simulations are capable of providing accurate qualitative and quantitative insights into enzyme catalysis in nonaqueous environments.

Chen and Arnold (Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5618−5622) have generated a 10 amino acid mutant (PC3) of subtilisin E (SE) that has enhanced activity in mixed DMF/water solvent systems. Through the use of molecular dynamics simulations on both SE and PC3 in water, DMF, and DMF/water (PC3 only) solvent systems, we have provided insights into how nonaqueous solvents affect protein structure and dynamics. On the basis of the observations reported herein, we propose that the PC3 mutant protein is more compatible with DMF as solvent than is the native SE protein. The concept of solvent compatibility embodies the ideas that in order for a protein to be active in organic solvents it must be able to retain its overall shape and that it must not become too rigid such that catalytic activity is compromised. Moreover, neither should the active site region be obstructed by conformational changes that block or structurally alter the active site nor should the active site binding pocket be blocked by solvent molecules (i.e., solvent inhibition). Attempts to determine how each individual amino acid substitution might cause these effects met with mixed results. Clearly, in the present case the individual mutations synergistically lead to the alteration in function through numerous subtle local changes in the structure and dynamics of the protein. Nonetheless, from the simulations, we were able to make some predictions regarding how a protein might be stabilized in a nonaqueous solvent environment.