NMR and X-ray Evidence That the HIV Protease Catalytic Aspartyl Groups Are Protonated in the Complex Formed by the Protease and a Non-Peptide Cyclic Urea-Based Inhibitor

Yamazaki, Toshimasa; Nicholson, Linda; Wingfield, Paul T.; Stahl, Stephen J.; Kaufman, Joshua D.; Eyermann, Charles J.; Hodge, C. Nicholas; Lam, Patrick Y.S.; Torchia, Dennis A.

doi:10.1021/ja00102a057

Cited by 138 publications

(174 citation statements)

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“…Using this approach, DMP323 was docked into the NMRderived structure of the protease (obtained without inclusion of the protease/DMP323 constraints) by placing the urea oxygen along the symmetry axis of the molecule and the inhibitor diol moieties proximal to the D25/D125 Asp carboxyl groups. This positioning of the DMP323 molecule is consistent with previous X-ray and NMR work (Grzesiek et al, 1994;Yamazaki et al, 1994aYamazaki et al, , 1994bNicholson et al, 1995b) on the interaction of DMP323 with the protease, and places the P,/Pj groups close to the amino acid residues in the complementary S j / S i sites of the protein (Wlodawer & Erickson, 1993). Positioning the inhibitor in this fashion allowed NOEs from several P,/P; sites and S,/S; sites to be distinguished.…”

Section: Tertiary Structure Of the Protease/dmp323 Complexsupporting

confidence: 92%

“…Although highresolution NMR techniques are well-suited to determine the structure of a molecule of the size of the protease, 22 kDa, the fact that the protein is subject to rapid autolysis frustrated solution studies until highly potent inhibitors, like the cyclic ureas, became available. Recent NMR studies of protease secondary structure (Yamazaki et al, 1994a), bound water, pK, values (Yamazaki et al, 1994b), and internal dynamics (Nicholson et al, 1995b) have provided novel information that complements the extensive structural information derived from crystal structures. We expect NMR structural studies to yield new insights about local conformation, particularly in regions of the protease that are disordered or have variable structures in the crystalline state.…”

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confidence: 99%

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Three‐dimensional solution structure of the HIV‐1 protease complexed with DMP323, a novel cyclic urea‐type inhibitor, determined by nuclear magnetic resonance spectroscopy

et al. 1996

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The three-dimensional solution structure of the HIV-I protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported. This is the first solution structure of an HIV proteasehnhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5", respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang C H et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A.As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.

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Section: Tertiary Structure Of the Protease/dmp323 Complexsupporting

confidence: 92%

mentioning

confidence: 99%

Three‐dimensional solution structure of the HIV‐1 protease complexed with DMP323, a novel cyclic urea‐type inhibitor, determined by nuclear magnetic resonance spectroscopy

et al. 1996

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“…Figure 7 shows the hydrogen bonds between residues from the active site and the inhibitor obtained after the final step of the computations on DMP-323 complex. It is notable that the predicted configuration of hydrogen bonds is exactly the same as suggested earlier from an analysis of the experimental data (Yamazaki et al 1994).…”

Section: Hiv-1 Proteasesupporting

confidence: 82%

“…Table 6 summarizes the results of the refinement of the network of hydrogen interactions obtained on a set of five neutron-diffraction structures crystallized at different pH. In the comparison of the hydrogen structure of individual residues we adopted a criterion used recently for similar purposes (Labute 2007): A prediction is assumed to be correct if the ionization states are the same, the tautomeric form or the orientation of flipping groups is the Experimental data from Yamazaki et al (1994), Wang et al (1996), Smith et al (1997), Velazquez-Campoy et al (2000). List of experimental data is the same as in Spassov et al (2001).…”

Section: Structural Optimization Of Hydrogen-bond Networkmentioning

confidence: 99%

A fast and accurate computational approach to protein ionization

Spassov¹,

Yan²

2008

Protein Science

137

130

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We report a very fast and accurate physics-based method to calculate pH-dependent electrostatic effects in protein molecules and to predict the pK values of individual sites of titration. In addition, a CHARMm-based algorithm is included to construct and refine the spatial coordinates of all hydrogen atoms at a given pH. The present method combines electrostatic energy calculations based on the Generalized Born approximation with an iterative mobile clustering approach to calculate the equilibria of proton binding to multiple titration sites in protein molecules. The use of the GBIM (Generalized Born with Implicit Membrane) CHARMm module makes it possible to model not only water-soluble proteins but membrane proteins as well. The method includes a novel algorithm for preliminary refinement of hydrogen coordinates. Another difference from existing approaches is that, instead of monopeptides, a set of relaxed pentapeptide structures are used as model compounds. Tests on a set of 24 proteins demonstrate the high accuracy of the method. On average, the RMSD between predicted and experimental pK values is close to 0.5 pK units on this data set, and the accuracy is achieved at very low computational cost. The pH-dependent assignment of hydrogen atoms also shows very good agreement with protonation states and hydrogen-bond network observed in neutron-diffraction structures. The method is implemented as a computational protocol in Accelrys Discovery Studio and provides a fast and easy way to study the effect of pH on many important mechanisms such as enzyme catalysis, ligand binding, protein-protein interactions, and protein stability.

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“…The catalytic aspartates were also modeled as protonated for the A76928 complex. pH-dependent changes in carbon chemical shifts suggest that Asp 25 and Asp 125 are protonated when DMP323 is bound to HIV protease (Yamazaki et al, 1994). A similar experiment showed shifts that are consistent with a monohydroxy inhibitor, KNI-272, binding to HIV protease with singly deprotonated catalytic aspartates (Wang et al, 1996b).…”

Section: Sirnuiation Protocolmentioning

confidence: 84%

Solvation studies of DMP323 and A76928 bound to HIV protease: Analysis of water sites using grand canonical Monte Carlo simulations

et al. 1998

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We examine the water solvation of the complex of the inhibitors DMP323 and A76928 bound to HIV-1 protease through grand canonical Monte Carlo simulations, and demonstrate the ability of this method to reproduce crystal waters and effectively predict water positions not seen in the DMP323 or A76928 structures. The simulation method is useful for identifying structurally important waters that may not be resolved in the crystal structures. It can also be used to identify water positions around a putative drug candidate docked into a binding pocket. Knowledge of these water positions may be useful in designing drugs to utilize them as bridging groups or displace them in the binding pocket. In addition, the method should be useful in finding water sites in homology models of enzymes for which crystal structures are unavailable.Keywords: grand canonical Monte Carlo; HIV protease; simulation; water Water molecules can play an important role in the binding affinity or specificity of an inhibitor. A prominent example is the structural water, WAT 301, seen in the crystal structure of nearly every HIV protease-inhibitor complex (Wlodawer & Erickson, 1993). WAT 301 bridges the backbone amide protons of Ile 50 and Ile 150 to inhibitor (and presumably substrate) carbonyls through hydrogen bonding. Mammalian protease-inhibitor complexes cannot accommodate it, so incorporation of a bidentate hydrogen-bond acceptor into an inhibitor to displace this water would achieve greater specificity for the viral protease over the mammalian proteases . A novel class of potent cyclic urea HIV protease inhibitors were rationally designed to displace WAT 301 to achieve greater specificity . This example clearly demonstrates the importance of the knowledge of water positions in a binding site for use in a structure-based drug design strategy.Because of the importance of water positions, several structural techniques such as NMR spectroscopy and X-ray crystallography

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NMR and X-ray Evidence That the HIV Protease Catalytic Aspartyl Groups Are Protonated in the Complex Formed by the Protease and a Non-Peptide Cyclic Urea-Based Inhibitor

Cited by 138 publications

References 1 publication

Three‐dimensional solution structure of the HIV‐1 protease complexed with DMP323, a novel cyclic urea‐type inhibitor, determined by nuclear magnetic resonance spectroscopy

Three‐dimensional solution structure of the HIV‐1 protease complexed with DMP323, a novel cyclic urea‐type inhibitor, determined by nuclear magnetic resonance spectroscopy

A fast and accurate computational approach to protein ionization

Solvation studies of DMP323 and A76928 bound to HIV protease: Analysis of water sites using grand canonical Monte Carlo simulations

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