Joshua D. Kaufman

Three‐dimensional solution structure of the 44 kDa ectodomain of SIV gp41

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e‐gp41), consisting of residues 27–149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e‐gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N‐terminal (residues 30–80) and C‐terminal (residues 107–147) helices connected by a 26 residue loop (residues 81–106). The N‐terminal helices of each subunit form a parallel coiled‐coil structure in the interior of the complex which is surrounded by the C‐terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non‐sequential intramolecular contacts. The helical core of SIV e‐gp41 is similar to recent X‐ray structures of truncated constructs of the helical core of HIV‐1 e‐gp41. The present structure establishes unambiguously the connectivity of the N‐ and C‐terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e‐gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e‐gp41 represents one of the largest protein structures determined by NMR to date.

Flexibility and function in HIV-1 protease

HIV protease is a homodimeric protein whose activity is essential to viral function. We have investigated the molecular dynamics of the HIV protease, thought to be important for proteinase function, bound to high affinity inhibitors using NMR techniques. Analysis of 15N spin relaxation parameters, of all but 13 backbone amide sites, reveals the presence of significant internal motions of the protein backbone. In particular, the flaps that cover the proteins active site of the protein have terminal loops that undergo large amplitude motions on the ps to ns time scale, while the tips of the flaps undergo a conformational exchange on the μs time scale. This enforces the idea that the flaps of the proteinase are flexible structures that facilitate function by permitting substrate access to and product release from the active site of the enzyme.

Biophysical and Functional Characterization of Full-length, Recombinant Human Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Produced in Escherichia coli COMPARISON OF WILD TYPE AND AMINO-TERMINAL ALANINE APPENDED VARIANT WITH IMPLICATIONS FOR THE MECHANISM OF TIMP FUNCTIONS

Matrix metalloproteinases (MMPs) function in the remodeling of the extracellular matrix that is integral for many normal and pathological processes. The tissue inhibitor of metalloproteinases family, including tissue inhibitor of metalloproteinases-2 (TIMP-2), regulates the activity of these multifunctional metalloproteinases. TIMP family members are proteinase inhibitors that contain six conserved disulfide bonds, one involving an amino-terminal cysteine residue that is critical for MMP inhibitor activity. TIMP-2 has been expressed in Escherichia coli, folded from insoluble protein, and functionally characterized. The wild type protein inhibited gelatinase A (MMP-2), whereas a variant with an alanine appended to the amino terminus (Ala+TIMP-2) was inactive. Removal of amino-terminal alanine by exopeptidase digestion restored protease inhibitor activity. This confirms the mechanistic importance of the amino-terminal amino group in the metalloproteinase inhibitory activity, as originally suggested from the x-ray structure of a complex of MMP-3 with TIMP-1 and a complex of TIMP-2 with MT-1-MMP. The Ala+TIMP-2 variant exhibited conformational, pro-MMP-2 complex formation and fibroblast growth modulating properties of the wild type protein. These findings demonstrate that Ala+TIMP-2 is an excellent biochemical tool for examining the specific role of MMP inhibition in the multiple functions ascribed to TIMPs.

Solution Structure of Anti-HIV-1 and Anti-Tumor Protein MAP30: Structural Insights into Its Multiple Functions

We present the solution structure of MAP30, a plant protein with anti-HIV and anti-tumor activities. Structural analysis and subsequent biochemical assays lead to several novel discoveries. First, MAP30 acts like a DNA glycosylase/apurinic (ap) lyase, an additional activity distinct from its known RNA N-glycosidase activity toward the 28S rRNA. Glycosylase/ap lyase activity explains MAP30s apparent inhibition of the HIV-1 integrase, MAP30s ability to irreversibly relax supercoiled DNA, and may be an alternative cytotoxic pathway that contributes to MAP30s anti-HIV/anti-tumor activities. Second, two distinct, but contiguous, subsites are responsible for MAP30s glycosylase/ap lyase activity. Third, Mn2+ and Zn2+ interact with negatively charged surfaces next to the catalytic sites, facilitating DNA substrate binding instead of directly participating in catalysis.

HIV-2 protease is inactivated after oxidation at the dimer interface and activity can be partly restored with methionine sulphoxide reductase.

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H(2)O(2) resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H(2)O(2) led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.

The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site.

BACKGROUND Hepatocyte growth factor (HGF) is a multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. The N-terminal (N) domain of HGF, containing a hairpin-loop region, is important for receptor binding and the potent biological activities of HGF. The N domain is also the primary binding site for heparin or heparan sulfate, which enhances, receptor/ligand oligomerization and modulates receptor-dependent mitogenesis. The rational design of artificial modulators of HGF signaling requires a detailed understanding of the structures of HGF and its receptor, as well as the role of heparin proteoglycan; this study represents the first step towards that goal. RESULTS We report here a high-resolution structure of the N domain of HGF. This first structure of HGF reveals a novel folding topology with a distinct pattern of charge distribution and indicates a possible heparin-binding site. CONCLUSIONS The hairpin-loop region of the N domain plays a major role in stabilizing the structure and contributes to a putative heparin-binding site, which explains why it is required for biological functions. These results suggest several basic and/or polar residues that may be important for use in further mutational studies of heparin binding.

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

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 sin4Δ 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.

Reversible Oxidative Modification as a Mechanism for Regulating Retroviral Protease Dimerization and Activation

ABSTRACT Human immunodeficiency virus protease activity can be regulated by reversible oxidation of a sulfur-containing amino acid at the dimer interface. We show here that oxidation of this amino acid in human immunodeficiency virus type 1 protease prevents dimer formation. Moreover, we show that human T-cell leukemia virus type 1 protease can be similarly regulated through reversible glutathionylation of its two conserved cysteine residues. Based on the known three-dimensional structures and multiple sequence alignments of retroviral proteases, it is predicted that the majority of retroviral proteases have sulfur-containing amino acids at the dimer interface. The regulation of protease activity by the modification of a sulfur-containing amino acid at the dimer interface may be a conserved mechanism among the majority of retroviruses.

Internal Dynamics of the Homotrimeric HIV‐1 Viral Coat Protein gp41 on Multiple Time Scales

Fusion of viral and cellular membranes is elicited by the HIV-1 envelope glycoprotein gp120/gp41. The precursor gp160, encoded by the Env gene, is cleaved post-translationally into two chains, gp120 and gp41, which remain non-covalently associated as a homotrimer of heterodimers and form a spike on the viral surface.[1] Upon CD4 recognition, the gp120 subunit dissociates from gp41, which remains anchored through its C-terminal transmembrane helix (TM) in the viral membrane. After gp120 dissociation, the N-terminal fusion peptide of gp41 is exposed and can insert into the host cell membrane.[2] Because of its high sequence conservation and its accessibility to the humoral immune system,[3] gp41 represents an attractive drug target for antiviral therapy as well as a key player in vaccine research.[4, 5]

Generation and characterization of a chimeric rabbit/human Fab for co-crystallization of HIV-1 Rev

Rev is a key regulatory protein of human immunodeficiency virus type 1. Its function is to bind to viral transcripts and effect export from the nucleus of unspliced mRNA, thereby allowing the synthesis of structural proteins. Despite its evident importance, the structure of Rev has remained unknown, primarily because Revs proclivity for polymerization and aggregation is an impediment to crystallization. Monoclonal antibody antigen-binding domains (Fabs) have proven useful for the co-crystallization of other refractory proteins. In the present study, a chimeric rabbit/human anti-Rev Fab was selected by phage display, expressed in a bacterial secretion system, and purified from the media. The Fab readily solubilized polymeric Rev. The resulting Fab/Rev complex was purified by metal ion affinity chromatography and characterized by analytical ultracentrifugation, which demonstrated monodispersity and indicated a 1:1 molar stoichiometry. The Fab binds with very high affinity, as determined by surface plasmon resonance, to a conformational epitope in the N-terminal half of Rev. The complex forms crystals suitable for structure determination. The ability to serve as a crystallization aid is a new application of broad utility for chimeric rabbit/human Fab. The corresponding single-chain antibody (scFv) was also prepared, offering the potential of intracellular antibody therapeutics against human immunodeficiency virus type 1.