G.M. Clore

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 structure of cyanovirin-N, a potent HIV-inactivating protein.

The solution structure of cyanovirin-N, a potent 11,000 M r HIV-inactivating protein that binds with high affinity and specificity to the HIV surface envelope protein gp120, has been solved by nuclear magnetic resonance spectroscopy, including extensive use of dipolar couplings which provide a priori long range structural information. Cyanovirin-N is an elongated, largely β-sheet protein that displays internal two-fold pseudosymmetry. The two sequence repeats (residues 1–50 and 51–101) share 32% sequence identity and superimpose with a backbone atomic root-mean-square difference of 1.3 Å. The two repeats, however, do not form separate domains since the overall fold is dependent on numerous contacts between them. Rather, two symmetrically related domains are formed by strand exchange between the two repeats. Analysis of surface hydrophobic clusters suggests the location of potential binding sites for protein–protein interactions.

A palindromic regulatory site within vertebrate GATA-1 promoters requires both zinc fingers of the GATA-1 DNA-binding domain for high-affinity interaction.

GATA-1, a transcription factor essential for the development of the erythroid lineage, contains two adjacent highly conserved zinc finger motifs. The carboxy-terminal finger is necessary and sufficient for specific binding to the consensus GATA recognition sequence: mutant proteins containing only the amino-terminal finger do not bind. Here we identify a DNA sequence (GATApal) for which the GATA-1 amino-terminal finger makes a critical contribution to the strength of binding. The site occurs in the GATA-1 gene promoters of chickens, mice, and humans but occurs very infrequently in other vertebrate genes known to be regulated by GATA proteins. GATApal is a palindromic site composed of one complete [(A/T)GATA(A/G)] and one partial (GAT) canonical motif. Deletion of the partial motif changes the site to a normal GATA site and also reduces by as much as eightfold the activity of the GATA-1 promoter in an erythroid precursor cell. We propose that GATApal is important for positive regulation of GATA-1 expression in erythroid cells.

Solution structure of the 40,000 Mr phosphoryl transfer complex between the N-terminal domain of enzyme I and HPr.

The solution structure of the first protein–protein complex of the bacterial phosphoenolpyruvate: sugar phosphotransferase system between the N–terminal domain of enzyme I (EIN) and the histidine–containing phosphocarrier protein HPr has been determined by NMR spectroscopy, including the use of residual dipolar couplings that provide long–range structural information. The complex between EIN and HPr is a classical example of surface complementarity, involving an essentially all helical interface, comprising helices 2, 2, 3 and 4 of the α–subdomain of EIN and helices 1 and 2 of HPr, that requires virtually no changes in conformation of the components relative to that in their respective free states. The specificity of the complex is dependent on the correct placement of both van der Waals and electrostatic contacts. The transition state can be formed with minimal changes in overall conformation, and is stabilized in favor of phosphorylated HPr, thereby accounting for the directionality of phosphoryl transfer.

Solution structure of the cellular factor BAF responsible for protecting retroviral DNA from autointegration.

The solution structure of the human barrier-to-autointegration factor, BAF, a 21,000 Mr dimer, has been solved by NMR, including extensive use of dipolar couplings which provide a priori long range structural information. BAF is a highly evolutionarily conserved DNA binding protein that is responsible for inhibiting autointegration of retroviral DNA, thereby promoting integration of retroviral DNA into the host chromosome. BAF is largely helical, and each subunit is composed of five helices. The dimer is elongated in shape and the dimer interface comprises principally hydrophobic contacts supplemented by a single salt bridge. Despite the absence of any sequence similarity to any other known protein family, the topology of helices 3–5 is similar to that of a number of DNA binding proteins, with helices 4 and 5 constituting a helix-turn-helix motif. A model for the interaction of BAF with DNA that is consistent with structural and mutagenesis data is proposed.

Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy

A 39-residue peptide (p7-DF) containing the two zinc binding domains of the p7 nucleocapsid protein was prepared by solid-phase peptide synthesis. The solution structure of the peptide was characterized using circular dichroic and nuclear magnetic resonance spectroscopy in both the presence and absence of zinc ions. Circular dichroic spectroscopy indicates that the peptide exhibits a random coil conformation in the absence of zinc but appears to form an ordered structure in the presence of zinc. Two-dimensional nuclear magnetic resonance spectroscopy indicates that the two zinc binding domains within the peptide form stable, but independent, units upon the addition of 2 equivalents of ZnCl2 per equivalent of peptide. Structure calculations on the basis of nuclear Overhauser (NOE) data indicate that the two zinc binding domains have the same polypeptide fold within the errors of the coordinates (approximately 0.5 A for the backbone atoms, the zinc atoms and the coordinating cysteine and histidine ligands). The linker region (Arg17-Gly23) is characterized by a very limited number of sequential NOEs and the absence of any non-sequential NOEs suggest that this region of polypeptide chain is highly flexible. The latter coupled with the occurrence of a large number of basic residues (four out of seven) in the linker region suggests that it may serve to allow adaptable positioning of the nucleic acid recognition sequences within the protein.

Preparation of Uniformly Isotope-labeled DNA Oligonucleotides for NMR Spectroscopy

Two methods for the large scale preparation of uniformly isotope-labeled DNA for NMR studies have been developed. The first method comprises the growth of a suitable plasmid harboring multiple copies of the desired oligonucleotide in a medium based on15N and 13C nutrients. The second method uses a polymerase chain reaction (PCR)-based approach with15N- and/or 13C-labeled deoxynucleoside triphosphates. The novelty of our PCR strategy over existing ones is that the primer and template are the identical molecule, resulting in an exponential growth in the length of the double strand that contains tandem repeats of the target DNA sequence. This novel PCR approach, which we have termed ESRA for endonuclease-sensitive repeat amplification, is easy to use, results in high yields, and can be accomplished at low costs. The utility of both methods is demonstrated for the preparation of a double-stranded 21-mer uniformly labeled with 15N and a double-stranded 17-mer DNA uniformly labeled with 15N and 13C.

SOLUTION STRUCTURE OF THE MU END DNA-BINDING IBETA SUBDOMAIN OF PHAGE MU TRANSPOSASE : MODULAR DNA RECOGNITION BY TWO TETHERED DOMAINS

The phage Mu transposase (MuA) binds to the ends of the Mu genome during the assembly of higher order nucleoprotein complexes. We investigate the structure and function of the MuA end‐binding domain (Iβγ). The three‐dimensional solution structure of the Iβ subdomain (residues 77–174) has been determined using multidimensional NMR spectroscopy. It comprises five α‐helices, including a helix–turn–helix (HTH) DNA‐binding motif formed by helices 3 and 4, and can be subdivided into two interacting structural elements. The structure has an elongated disc‐like appearance from which protrudes the recognition helix of the HTH motif. The topology of helices 2–4 is very similar to that of helices 1–3 of the previously determined solution structure of the MuA Iγ subdomain and to that of the homeodomain family of HTH DNA‐binding proteins. We show that each of the two subdomains binds to one half of the 22 bp recognition sequence, Iβ to the more conserved Mu end distal half (β subsite) and Iγ to the Mu end proximal half (γ subsite) of the consensus Mu end‐binding site. The complete Iβγ domain binds the recognition sequence with a 100‐ to 1000‐fold higher affinity than the two subdomains independently, indicating a cooperative effect. Our results show that the Mu end DNA‐binding domain of MuA has a modular organization, with each module acting on a specific part of the 22 bp binding site. Based on the present binding data and the structures of the Iβ and Iγ subdomains, a model for the interaction of the complete Iβγ domain with DNA is proposed.

Understanding SRY-Related 46X,Y Sex Reversal at the Atomic Level

There is considerable evidence that the Y chromosome encoded testis determining factor, known as SRY, constitutes the primary active binary switch which regulates the transcription of a cascade of genes which in turn direct the development of the primordial gonad into the male testes (Goodfellow and Lovell-Badge 1993; McElreavey et al. 1993; Gustafson and Donahoe 1994; Haqq et al. 1994; Werner et al. 1996a). In the absence of functional SRY, either through mutations in SRY or because of chromosomal makeup (viz. XX females), the primordial gonad develops into ovaries. One potential target for SRY is the promoter for the gene encoding the Mullerian inhibtory substance (MIS) whose product is responsible for the regression of the female Mullerian ducts.

Zinc Fingers Involved in MHC Class I Gene Regulation: Use of Synthetic Peptides for Structural Analysis

Two zinc finger proteins capable of binding specific DNA sequence motifs in the regulatory region of the MHC Class I genes have been described. The protein binding to the DNA sequence TGGGGATTCCCCAT contains two zinc finger motifs of the TFIIIA like sequence. Peptides comprising the double and the two individual finger regions have been prepared by solid phase peptide synthesis. Spectral studies have shown that the peptides have metal binding capabilities and that binding of the metal results in specific conformational changes. Binding studies showed that stable DNA-peptide complexes are formed with the double fingers. NMR results clearly demonstrate that single fingers are capable of forming well-defined three-dimensional structures.