Michael F. Summers

Structure of the Amino-Terminal Core Domain of the HIV-1 Capsid Protein

The three-dimensional structure of the amino-terminal core domain (residues 1 through 151) of the human immunodeficiency virus-type 1 (HIV-1) capsid protein has been solved by multidimensional heteronuclear magnetic resonance spectroscopy. The structure is unlike those of previously characterized viral coat proteins and is composed of seven α helices, two β hairpins, and an exposed partially ordered loop. The domain is shaped like an arrowhead, with the β hairpins and loop exposed at the trailing edge and the carboxyl-terminal helix projecting from the tip. The proline residue Pro1 forms a salt bridge with a conserved, buried aspartate residue (Asp51), which suggests that the amino terminus of the protein rearranges upon proteolytic maturation. The binding site for cyclophilin A, a cellular rotamase that is packaged into the HIV-1 virion, is located on the exposed loop and encompasses the essential proline residue Pro90. In the free monomeric domain, Pro90 adopts kinetically trapped cis and trans conformations, raising the possibility that cyclophilin A catalyzes interconversion of the cis- and trans-Pro90 loop structures.

Crystal structure of dimeric HIV-1 capsid protein.

X-ray diffraction analysis of a human immunodeficiency virus (HIV-1) capsid (CA) protein shows that each monomer within the dimer consists of seven α-helices, five of which are arranged in a coiled coil-like structure. Sequence assignments were made for two of the helices, and tentative connectivity of the remainder of the protein was confirmed by the recent solution structure of a monomeric N-terminal fragment. The C-terminal third of the protein is mostly disordered in the crystal. The longest helices in the coiled coil-like structure are separated by a long, highly antigenic peptide that includes the binding site of an antibody fragment complexed with CA in the crystal. The site of binding of the Fab, the position of the antigenic loop and the site of cleavage between the matrix protein and CA establish the side of the dimer that would be on the exterior of the retroviral core.

How retroviruses select their genomes

As retroviruses assemble in infected cells, two copies of their full-length, unspliced RNA genomes are selected for packaging from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Understanding the molecular details of genome packaging is important for the development of new antiviral strategies and to enhance the efficacy of retroviral vectors used in human gene therapy. Recent studies of viral RNA structure in vitro and in vivo and high-resolution studies of RNA fragments and protein–RNA complexes are helping to unravel the mechanism of genome packaging and providing the first glimpses of the initial stages of retrovirus assembly.

Organocobalt B12 models: axial ligand effects on the structural and coordination chemistry of cobaloximes

Article de synthese presentant les structures cristallines et moleculaires de molecules contenant la moitie Co(DH) 2 , ou DH est le monoanion de la dimethylglyoxime. Les structures RX sont comparees aux structures en solution. Discussion des implications pour la biochimie de la vitamine B 12

113Cd NMR spectroscopy of coordination compounds and proteins

Abstract Despite the proven utility of 113Cd NMR chemical shifts for qualitatively or, in some cases, semiquantitatively assessing the nature of ligands at metal binding sites in metalloproteins and for studying enzyme function and mechanisms, it is likely that the full potential of 113Cd spectroscopy as a metallobioprobe has yet to be realized. The development of a more quantitative relationship between structural parameters and 113Cd chemical shift has been hampered in part by the lack of suitable model compounds and chemical dynamics [6]. As described throughout this review, these challenges are currently being met through applications of solid-state and low-temperature solution NMR methods and the design and study of less labile protein model compounds. However, even for cases where dynamics and solvent or anion effects can be ruled out, it appears that ligand atom type and number, bond lengths, coordination geometry, and neighboring atoms (including other metals) all influence 113Cd shifts. Studies of coordination compounds in solution have thus far provided useful information on the influence of donor-atom type and number on isotropic Cd chemical shifts. The influence on shift of coordination geometry and CdL bond lengths will probably only be adequately evaluated using the single crystal approach pioneered by Ellis and co-workers. Studies of the Cd ion bound exclusively by O-donor ligands will likely also be only adequately addressed through solid-state NMR methods due to lability problems with these ligands. In this area, model compounds with O-donor ligands which exhibit the unusual high-field shifts found for ICaBP and HRP are lacking. 113Cd NMR chemical shift information alone is at present inadequate for unambiguous determination of structural features or structural changes at the metal binding sites of metalloproteins. In this regard, HMQC NMR spectroscopy promises to be a particularly useful tool for identifying the ligands which are coordinated to Cd. For cases where ligand exchange or nuclear relaxation rates are high (relative to 1/J), however, this method will not work and determination of structural features may rely exclusively on chemical shift analyses. It is therefore important that both techniques be developed and utilized.

Structure of the N-terminal 283-residue fragment of the immature HIV-1 Gag polyprotein.

The capsid protein (CA) of the mature human immunodeficiency virus (HIV) contains an N-terminal β-hairpin that is essential for formation of the capsid core particle. CA is generated by proteolytic cleavage of the Gag precursor polyprotein during viral maturation. We have determined the NMR structure of a 283-residue N-terminal fragment of immature HIV-1 Gag (Gag283), which includes the intact matrix (MA) and N-terminal capsid (CAN) domains. The β-hairpin is unfolded in Gag283, consistent with the proposal that hairpin formation occurs subsequent to proteolytic cleavage of Gag, triggering capsid assembly. Comparison of the immature and mature CAN structures reveals that β-hairpin formation induces a ∼2 Å displacement of helix 6 and a concomitant displacement of the cyclophylin-A (CypA)-binding loop, suggesting a possible allosteric mechanism for CypA-mediated destabilization of the capsid particle during infectivity.

Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus.

All retroviruses specifically package two copies of their genomes during virus assembly, a requirement for strand-transfer-mediated recombination during reverse transcription. Genomic RNA exists in virions as dimers, and the overlap of RNA elements that promote dimerization and encapsidation suggests that these processes may be coupled. Both processes are mediated by the nucleocapsid domain (NC) of the retroviral Gag polyprotein. Here we show that dimerization-induced register shifts in base pairing within the Ψ-RNA packaging signal of Moloney murine leukaemia virus (MoMuLV) expose conserved UCUG elements that bind NC with high affinity (dissociation constant = 75 ± 12 nM). These elements are base-paired and do not bind NC in the monomeric RNA. The structure of the NC complex with a 101-nucleotide ‘core encapsidation’ segment of the MoMuLV Ψ site reveals a network of interactions that promote sequence- and structure-specific binding by NCs single CCHC zinc knuckle. Our findings support a structural RNA switch mechanism for genome encapsidation, in which protein binding sites are sequestered by base pairing in the monomeric RNA and become exposed upon dimerization to promote packaging of a diploid genome.

Structure of the carboxy-terminal LIM domain from the cysteine rich protein CRP

The three dimensional solution structure of the carboxy terminal LIM domain of the avian Cysteine Rich Protein (CRP) has been determined by nuclear magnetic resonance spectroscopy. The domain contains two zinc atoms bound independently in CCHC (C=Cys, H=His) and CCCC modules. Both modules contain two orthogonally-arranged antiparallel β-sheets, and the CCCC module contains an α-helix at its C terminus. The modules pack due to hydrophobic interactions forming a novel global fold. The structure of the C-terminal CCCC module is essentially identical to that observed for the DNA-interactive CCCC modules of the GATA-1 and steroid hormone receptor DNA binding domains, raising the possibility that the LIM motif may have a DNA binding function.

NMR Detection of Structures in the HIV-1 5′-Leader RNA That Regulate Genome Packaging

An RNA structural switch regulates whether the HIV genome is translated or dimerized and packaged. The 5′-leader of the HIV-1 genome regulates multiple functions during viral replication via mechanisms that have yet to be established. We developed a nuclear magnetic resonance approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the unique-5′ region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin and enhances binding by the nucleocapsid (NC) protein, which is the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.

Structure of the HIV-1 RNA packaging signal

Structural signals that direct HIV packaging During the viral replication cycle of HIV, unspliced dimeric RNA genomes are efficiently packaged into new virions at the host cell membrane. Packaging is directed by a region at the start of the genome, the 5′ leader. The architecture of the 5′ leader remains controversial. Keane et al. developed nuclear magnetic resonance methods to determine the structure of a 155-nucleotide-long region of the 5′ leader that can direct viral packaging. The structure shows how the 5′ leader binds to the HIV protein that directs packaging, how unspliced dimeric genomes are selected for packaging, and how translation is suppressed when the genome dimerizes. Science, this issue p. 917 A nuclear magnetic resonance structure of a region of the HIV-1 RNA 5′ leader gives insight into how the viral genome is selected for packaging. The 5′ leader of the HIV-1 genome contains conserved elements that direct selective packaging of the unspliced, dimeric viral RNA into assembling particles. By using a 2H-edited nuclear magnetic resonance (NMR) approach, we determined the structure of a 155-nucleotide region of the leader that is independently capable of directing packaging (core encapsidation signal; ΨCES). The RNA adopts an unexpected tandem three-way junction structure, in which residues of the major splice donor and translation initiation sites are sequestered by long-range base pairing and guanosines essential for both packaging and high-affinity binding to the cognate Gag protein are exposed in helical junctions. The structure reveals how translation is attenuated, Gag binding promoted, and unspliced dimeric genomes selected, by the RNA conformer that directs packaging.