Stephen J. Stahl

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.

Solution NMR of proteins within polyacrylamide gels: Diffusional properties and residual alignment by mechanical stress or embedding of oriented purple membranes

The diffusive properties of biomacromolecules within the aqueous phase of polyacrylamide gels are described. High quality NMR spectra can be obtained under such conditions. As compared to water, a fivefold reduction in the translational diffusion constant, but only a 1.6-fold decrease (1.4-fold increase) in amide-15N T2 (T1) are observed for human ubiquitin within a 10% acrylamide gel. Weak alignment of the solute macromolecules can be achieved within such gels by vertical or radial compression or by the embedding of magnetically oriented purple membrane fragments. The methods are applied to derive residual dipolar couplings for human HIV-1 Nef and ubiquitin.

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.

Anisotropic rotational diffusion of perdeuterated HIV protease from 15N NMR relaxation measurements at two magnetic fields

Summary15N NMR relaxation times in perdeuterated HIV-1 protease, complexed with the sub-nanomolar inhibitor DMP323, have been measured at 600 and 360 MHz 1H frequency. The relative magnitudes of the principal components of the inertia tensor, calculated from the X-ray coordinates of the protein-drug complex, are 1.0:0.85:0.44. The relation between the T1/T2 ratios observed for the individual backbone amides and their N-H orientation within the 3D structure of the protease dimer yields a rotational diffusion tensor oriented nearly collinear to the inertia tensor. The relative magnitudes of its principal components (1.00:1.11:1.42) are also in good agreement with hydrodynamic modeling results. The orientation and magnitude of the diffusion tensors derived from relaxation data obtained at 360 and 600 MHz are nearly identical. The anisotropic nature of the rotational diffusion has little influence on the order parameters derived from the 15N T1 and T2 relaxation times; however, if anisotropy is ignored, this can result in erroneous identification of either exchange broadening or internal motions on a nanosecond time scale. The average ratio of the T1 values measured at 360 and 600 MHz is 0.50±0.015, which is slightly larger than the value of 0.466 expected for an isotropic rigid rotor with τc = 10.7 ns. The average ratio of the T2 values measured at 360 and 600 MHz is 1.14±0.04, which is also slightly larger than the expected ratio of 1.11. This magnetic field dependence of the T1 and T2 relaxation times suggests that the spectral density contribution from fast internal motions is not negligible, and that the chemical shift anisotropy of peptide backbone amides, on average, is larger than the 160 ppm value commonly used in 15N relaxation studies of proteins.

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.

Implications of the HIV-1 Rev Dimer Structure at 3. 2 A Resolution for Multimeric Binding to the Rev Response Element.

HIV-1 Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process Rev oligomerizes in association with a highly structured RNA motif, the Rev response element. Crystallographic studies of Rev have been hampered by the protein’s tendency to aggregate, but Rev has now been found to form a stable soluble equimolar complex with a specifically engineered monoclonal Fab fragment. We have determined the structure of this complex at 3.2 Å resolution. It reveals a molecular dimer of Rev, bound on either side by a Fab, where the ordered portion of each Rev monomer (residues 9–65) contains two coplanar α-helices arranged in hairpin fashion. Subunits dimerize through overlapping of the hairpin prongs. Mating of hydrophobic patches on the outer surface of the dimer is likely to promote higher order interactions, suggesting a model for Rev oligomerization onto the viral RNA.

Structure, Assembly, and Antigenicity of Hepatitis B Virus Capsid Proteins

Publisher Summary This chapter reviews current information pertaining to the structure and assembly properties of hepatitis B virus (HBV) capsid protein, as well as the insights into its antigenicity and other interactions. HBV has a small (3.2 kb) DNA genome, although this modest genetic endowment is amplified by a variety of strategies, including alternative expression products of the same gene. In the replication cycle of HBV, the genome is initially incorporated into the assembling virus particle as a single-stranded RNA molecule—the pregenome—that is subsequently retrotranscribed in situ by the viral reverse transcriptase (RT). The DNA-containing nucleocapsid subsequently becomes enveloped by a membrane containing the viral glycoprotein—surface antigen (sAg), of which there are three size variants called S, M, and L, respectively—to yield the completely assembled and infectious virion. The capsid protein of HBV has several unexpected properties. It was found to have a novel fold, rich in a helix, and quite distinct from the eight-stranded b barrel that was common to the first dozen or so capsid proteins to be solved (from plant, animal, and bacterial viruses) and the other capsid protein folds that have been determined more recently.

The morphogenic linker peptide of HBV capsid protein forms a mobile array on the interior surface.

Many capsid proteins have peptides that influence their assembly. In hepatitis B virus capsid protein, the peptide STLPETTVV, linking the shell‐forming ‘core’ domain and the nucleic acid‐binding ‘protamine’ domain, has such a role. We have studied its morphogenic properties by permuting its sequence, substituting it with an extraneous peptide, deleting it to directly fuse the core and protamine domains and assembling core domain dimers with added linker peptides. The peptide was found to be necessary for the assembly of protamine domain‐containing capsids, although its size‐determining effect tolerates some modifications. Although largely invisible in a capsid crystal structure, we could visualize linker peptides by cryo‐EM difference imaging: they emerge on the inner surface and extend from the capsid protein dimer interface towards the adjacent symmetry axis. A closely sequence‐similar peptide in cellobiose dehydrogenase, which has an extended conformation, offers a plausible prototype. We propose that linker peptides are attached to the capsid inner surface as hinged struts, forming a mobile array, an arrangement with implications for morphogenesis and the management of encapsidated nucleic acid.

Diversity of core antigen epitopes of hepatitis B virus

Core antigen (cAg), the viral capsid, is one of the three major clinical antigens of hepatitis B virus. cAg has been described as presenting either one or two conformational epitopes involving the “immunodominant loop.” We have investigated cAg antigenicity by cryo-electron microscopy at ≈11-Å resolution of capsids labeled with monoclonal Fabs, combined with molecular modeling, and describe here two conformational epitopes. Both Fabs bind to the dimeric external spikes, and each epitope has contributions from the loops on both subunits, explaining their discontinuous nature: however, their binding aspects and epitopes differ markedly. To date, four cAg epitopes have been characterized: all are distinct. Although only two regions of the capsid surface are accessible to antibodies, local clustering of the limited number of exposed peptide loops generates a potentially extensive set of discontinuous epitopes. This diversity has not been evident from competition experiments because of steric interference effects. These observations suggest an explanation for the distinction between cAg and e-antigen (an unassembled form of capsid protein) and an approach to immunodiagnosis, exploiting the diversity of cAg epitopes.

Characterization of a Conformational Epitope on Hepatitis B Virus Core Antigen and Quasiequivalent Variations in Antibody Binding

ABSTRACT We have characterized a conformational epitope on capsids of hepatitis B virus (HBV) by cryo-electron microscopy and three-dimensional image reconstruction of Fab-labeled capsids to ∼10-Å resolution, combined with molecular modeling. The epitope straddles the interface between two adjacent subunits and is discontinuous, consisting of five peptides—two on one subunit and three on its neighbor. Together, the two icosahedral forms of the HBV capsid—T=3 and T=4 particles—present seven quasiequivalent variants of the epitope. Of these, only three bind this Fab. Occupancy ranges from ∼100 to ∼0%, reflecting conformational variations in the epitope and steric blocking effects. In the former, small shifts of the component peptides have large effects on binding affinity. This approach appears to hold general promise for elucidating conformational epitopes of HBV and other viruses, including those of neutralizing and diagnostic significance.