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

Yamazaki, Toshimasa; Hinck, Andrew P.; Wang, Yun Xing; Nicholson, Linda; Torchia, Dennis A.; Wingfield, Paul T.; Stahl, Stephen J.; Kaufman, Joshua D.; Chang, C. H.; Domaille, Peter J.; Lam, Patrick Y.S.

doi:10.1002/pro.5560050311

Cited by 76 publications

(79 citation statements)

References 36 publications

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“…Gal11 KIX and Gcn4 Expression and Purification for NMR Studies-Expression in E. coli of isotopically labeled proteins ( 13 C, 15 N and 2 H, 15 N) was performed as described previously (54). After cell breakage, the His 6 -tagged fusion proteins were mostly in the soluble cell fraction, but varying amounts of denaturants were included during purification to prevent nonspecific protein interactions with host proteins.…”

Section: Methodsmentioning

confidence: 99%

Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo

Jedidi

Zhang

Qiu

et al. 2010

Journal of Biological Chemistry

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Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4⌬ cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

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Section: Methodsmentioning

confidence: 99%

Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo

Jedidi

Zhang

Qiu

et al. 2010

Journal of Biological Chemistry

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“…For two of these proteins, HIV-1 protease and maltose binding protein (MBP), the overall rotational diffusion had been characterized experimentally. For HIV-1 protease we used the NMR solution structure published by Yamazaki et al 29 (PDB code 1BVG) and the components of the rotational diffusion tensor (Table 1) determined by Tjandra et al 30 For MBP we used the NMR structure derived by Mueller et al 31 (PDB code 1EZP). The orientation of MBP's rotational diffusion tensor with respect to the molecular reference frame of the protein was not available from the published 15 N relaxation study.3 Thus, to obtain a complete overall diffusion tensor of MBP we reanalyzed raw NMR relaxation data from that paper3 (see Supporting Information).…”

Section: Methodsmentioning

confidence: 99%

Structural Assembly of Multidomain Proteins and Protein Complexes Guided by the Overall Rotational Diffusion Tensor

Ryabov

Fushman

2007

J. Am. Chem. Soc.

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We present a simple and robust approach that uses the overall rotational diffusion tensor as a structural constraint for domain positioning in multidomain proteins and protein-protein complexes. This method offers the possibility to use NMR relaxation data for detailed structure characterization of such systems provided the structures of individual domains are available. The proposed approach extends the concept of using long-range information contained in the overall rotational diffusion tensor. In contrast to the existing approaches, we use both the principal axes and principal values of protein's rotational diffusion tensor to determine not only the orientation but also the relative positioning of the individual domains in a protein. This is achieved by finding the domain arrangement in a molecule that provides the best possible agreement with all components of the overall rotational diffusion tensor derived from experimental data. The accuracy of the proposed approach is demonstrated for two protein systems with known domain arrangement and parameters of the overall tumbling: the HIV-1 protease homodimer and Maltose Binding Protein. The accuracy of the method and its sensitivity to domain positioning is also tested using computer-generated data for three protein complexes, for which the experimental diffusion tensors are not available. In addition, the proposed method is applied here to determine, for the first time, the structure of both open and closed conformations of Lys48-linked di-ubiquitin chain, where domain motions render impossible accurate structure determination by other methods. The proposed method opens new avenues for improving structure characterization of proteins in solution.

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“…The mutant proteases used in the study are listed in Fig. 1 (16). All proteins were isolated using a common protocol, which comprises isolation of inclusion bodies followed by fractionation of the protease by size-exclusion and reversed-phase high-pressure liquid chromatography.…”

Section: Methodsmentioning

confidence: 99%

Folded Monomer of HIV-1 Protease

Ishima¹,

Ghirlando²,

Tőzsér³

et al. 2001

Journal of Biological Chemistry

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133

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The mature human immunodeficiency virus type 1 protease rapidly folds into an enzymatically active stable dimer, exhibiting an intricate interplay between structure formation and dimerization. We now show by NMR and sedimentation equilibrium studies that a mutant protease containing the R87K substitution (PR R87K ) within the highly conserved Gly 86 -Arg 87 -Asn 88 sequence forms a monomer with a fold similar to a single subunit of the dimer. However, binding of the inhibitor DMP323 to PR R87K produces a stable dimer complex. Based on the crystal structure and our NMR results, we postulate that loss of specific interactions involving the side chain of Arg 87 destabilizes PR R87K by perturbing the inner C-terminal ␤-sheet (residues 96 -99 from each monomer), a region that is sandwiched between the two ␤-strands formed by the N-terminal residues (residues 1-4) in the mature protease. We systematically examined the folding, dimerization, and catalytic activities of mutant proteases comprising deletions of either one of the terminal regions (residues 1-4 or 96 -99) or both. Although both N-and C-terminal ␤-strands were found to contribute to dimer stability, our results indicate that the inner Cterminal strands are absolutely essential for dimer formation. Knowledge of the monomer fold and regions critical for dimerization may aid in the rational design of novel inhibitors of the protease to overcome the problem of drug resistance. Human immunodeficiency virus type 1 (HIV-1)1 is a 99-residue aspartic acid protease and is active as a homodimer. The interface of the free protease dimer is stabilized through interactions between the two subunits at the active site and at the termini (1, 2). The protease catalyzes its own release from the Gag-Pol polyprotein in addition to the maturation of the virally encoded structural proteins and replication enzymes required for the assembly and production of viable virions (3). Thus, the protease has served as one of the primary targets for the development of drugs against AIDS. Structure-based design of drugs targeted against the wild-type mature protease has aided in the development of several potent inhibitors that are specific for binding to the active site (4). Although several of these drugs are in clinical use and have curtailed the progression of the disease, the effectiveness of long-term treatment has been restricted due to naturally selected protease variants exhibiting lower affinity for the drugs than the wild-type enzyme. Various drug-resistant mutants of the protease have been identified (5). Different resistance mechanisms based on the observed structural changes in drug-resistant mutants have been proposed. In general, the mutants modulate structure and interactions within the active site as well as inter-and intrasubunit flexibility (6 -8).Because of the difficulty in designing active-site inhibitors that avoid the problem of drug resistance, the target region for drug development has been extended to areas in which no drug-selected mutations have occurred to date...

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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

Cited by 76 publications

References 36 publications

Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo

Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo

Structural Assembly of Multidomain Proteins and Protein Complexes Guided by the Overall Rotational Diffusion Tensor

Folded Monomer of HIV-1 Protease

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