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Biomedical subjects

S C Harrison

Publications and source records attributed to S C Harrison.

At least 19 recordsLinked to original sources
PubMed1992

The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex.

The yeast transcriptional activator GCN4 is 1 of over 30 identified eukaryotic proteins containing the basic region leucine zipper (bZIP) DNA-binding motif. We have determined the crystal structure of the GCN4 bZIP element complexed with DNA at 2.9 A resolution. The bZIP dimer is a pair of continuous alpha helices that form a parallel coiled coil over their carboxy-terminal 30 residues and gradually diverge toward their amino termini to pass through the major groove of the DNA-binding site. The coiled-coil dimerization interface is oriented almost perpendicular to the DNA axis, giving the complex the appearance of the letter T. There are no kinks or sharp bends in either bZIP monomer. Numerous contacts to DNA bases and phosphate oxygens are made by basic region residues that are conserved in the bZIP protein family. The details of the bZIP dimer interaction with DNA can explain recognition of the AP-1 site by the GCN4 protein.

Amino Acid Sequence↗
PubMed1992

Human immunodeficiency virus gp120 binding C'C" ridge of CD4 domain 1 is also involved in interaction with class II major histocompatibility complex molecules.

Using site-directed mutagenesis informed by high-resolution CD4 structural data, we have investigated the role of residues of the C'C'' ridge region of human CD4 on class II major histocompatibility complex (MHC) binding. This C'C'' ridge is homologous to the CDR2 loop of an immunoglobulin variable domain and is known to contain the binding site for human immunodeficiency virus (HIV) coat glycoprotein gp120. Here we report that this region is also involved in interaction with class II MHC. Exposed positively charged residues Lys-35, Lys-46, and Arg-59 and the exposed hydrophobic residue Phe-43 contribute significantly to class II MHC binding. Moreover, mutations in the buried residues Trp-62 and Ser-49, which support the top and bottom of the C'C'' ridge, respectively, disrupt class II MHC interaction. The HIV binding region appears to involve a restricted area of the larger class II MHC binding site on CD4. Strategies of drug design aimed at interrupting CD4-HIV interaction will need to consider the extensive overlap between class II MHC and HIV gp120 binding surfaces in this region of CD4.

Animals↗
PubMed1992

The human immunodeficiency virus gp120 binding site on CD4: delineation by quantitative equilibrium and kinetic binding studies of mutants in conjunction with a high-resolution CD4 atomic structure.

The first immunoglobulin V-like domain of CD4 contains the binding site for human immunodeficiency virus gp120. Guided by the atomic structure of a two-domain CD4 fragment, we have examined gp120 interaction with informative CD4 mutants, both by equilibrium and kinetic analysis. The binding site on CD4 appears to be a surface region of about 900 A2 on the C" edge of the domain. It contains an exposed hydrophobic residue, Phe43, on the C" strand and four positively charged residues, Lys29, Lys35, Lys46, and Arg59, on the C, C', C", and D strands, respectively. Replacement of Phe43 with Ala or Ile reduces affinity for gp120 by more than 500-fold; Tyr, Trp, and Leu substitutions have smaller effects. The four positively charged side chains each make significant contributions (7-50-fold). This CD4 site may dock into a conserved hydrophobic pocket bordered by several negatively charged residues in gp120. Class II major histocompatibility complex binding includes the same region on CD4; this overlap needs to be considered in the design of inhibitors of the CD4-gp120 interaction.

Antibodies, Monoclonal↗
PubMed1992

Crystallization of Sindbis virus and its nucleocapsid.

Crystals of Sindbis virus, which contains a lipid-bilayer membrane, have been grown using polyethylene glycol. The space group is R32, a = b = 640 A, c = 1520 A. The crystals are highly mosaic, and recorded diffraction is therefore restricted to spacings of about 30 A. The crystals show that the packing of glycoproteins E1 and E2 in the icosahedral outer shell is sufficiently precise that it permits regular and repeated interactions between virus particles in the lattice. Crystals of Sindbis nucleocapsids have also been grown. The limited diffraction data are consistent with close packing of nucleocapsids 404 A in diameter.

Capsid↗
PubMed1992

DNA recognition by GAL4: structure of a protein-DNA complex.

A specific DNA complex of the 65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4, has been analysed at 2.7 A resolution by X-ray crystallography. The protein binds as a dimer to a symmetrical 17-base-pair sequence. A small, Zn(2+)-containing domain recognizes a conserved CCG triplet at each end of the site through direct contacts with the major groove. A short coiled-coil dimerization element imposes 2-fold symmetry. A segment of extended polypeptide chain links the metal-binding module to the dimerization element and specifies the length of the site. The relatively open structure of the complex would allow another protein to bind coordinately with GAL4.

Amino Acid Sequence↗
PubMed1992

Solution structure of the DNA-binding domain of Cd2-GAL4 from S. cerevisiae.

The GAL4 protein activates transcription of the genes required for galactose utilization in Saccharomyces cerevisiae. The protein, consisting of 881 amino acids, is dimeric when bound to one of the approximately twofold symmetrical DNA sites present in the galactose upstream activating sequence (UASG). Here we use two-dimensional NMR spectroscopy to determine the structure of an amino-terminal fragment of GAL4 (residues 1-65). This fragment, a monomer in solution, binds as a dimer specifically to UASG-containing DNA. Residues 9-40 form a well defined, compact globular cluster, whereas residues 1-8 and 41-66 show considerable conformational mobility in the absence of DNA. The compact domain contains a motif in which six cysteines, located on two symmetrically related helix/extended strand units connected by a long loop, coordinate two central zinc ions, forming a bimetal-thiolate cluster. The zincs were replaced by NMR-active 113Cd in most of our work and structural parameters are therefore derived from the Cd2-protein. The structure obtained for the GAL4 DNA-binding domain represents a novel DNA-binding motif. Essentially the same conformation is observed for the compact domain in solution using NMR techniques as was seen for the central core of the N-terminal fragment bound to DNA using crystallographic techniques. Thus, the core of the DNA-binding domain changes little upon binding DNA.

Amino Acid Sequence↗
PubMed1991

Structure of simian virus 40 at 3.8-A resolution.

The crystallographically determined structure of simian virus 40 shows that the 72 pentamers of viral protein VP1, which form the outer shell, have identical conformations except for the C-terminal arms of their subunits. Five arms emerge from each pentamer and insert into neighbouring pentamers. This tying together of standard building blocks allows for the required variability in packing geometry without sacrificing specificity.

Amino Acid Sequence↗
PubMed1991

The phage 434 Cro/OR1 complex at 2.5 A resolution.

The crystal structure of phage 434 Cro protein in complex with a 20 base-pair DNA fragment has been determined to 2.5 A resolution. The DNA fragment contains the sequence of the OR1 operator site. The structure shows a bent conformation for the DNA, straighter at the center and more bent at the ends. The central base-pairs adopt conformations with significant deviations from coplanarity. The two molecules interact extensively along their common interface, both through hydrogen bonds and van der Waals interactions. The significance of these interactions for operator binding and recognition is discussed.

Amino Acid Sequence↗
PubMed1991

Crystallization and X-ray diffraction studies of a soluble form of the human transferrin receptor.

A soluble tryptic fragment of the human transferrin receptor (residues 121 to 760) has been crystallized from 2.8 M-KCl (pH 6.2) and polyethylene glycol 8000. This fragment retains the transferrin-binding activity of intact transferrin receptor. Although the trypsin treatment removes the intermolecular disulfide bonds, the receptor fragment is dimeric both under physiological conditions and at the high salt concentrations used for crystallization. The receptor fragment crystallizes in the orthorhombic space group P2(1)2(1)2(1), a = 105.5 A, b = 224.5 A, c = 363.5 A. The crystals are extremely radiation sensitive. Their diffraction extends to 3.8 A, and there is some diffuse scatter with helical characteristics. Analysis of these diffraction patterns indicates that the transferrin receptor fragments are arranged in continuous 8-fold symmetric helical columns parallel to the c axis, with a total of 32 receptor fragment monomers in the unit cell. A structure determination is in progress.

Humans↗
PubMed1991

Prospective study of quality of life before and after coronary artery bypass grafting.

OBJECTIVES: Measurement of changes in patients' perceptions of how differing states of health affect their lives and determination of the ability of preoperative variables to predict outcome after coronary artery bypass grafting. DESIGN: Prospective study with completion of questionnaires before coronary artery bypass grafting and at three months, one year, and five years afterwards. SETTING: Regional cardiothoracic centre. PATIENTS: 100 Male patients all aged below 60 at the time of operation, who were patients of two cardiothoracic surgeons. MAIN OUTCOME MEASURES: Patients' assessment of their health state in terms of functional capacity and aspects of distress, according to the Nottingham health profile and outcome of operation in terms of changes in symptoms, working life, and daily activities determined by self completed study questionnaires before operation and at three and six months afterwards. RESULTS: Intermediate one year results are reported. The differences between the Nottingham health profile scores before operation and at three months afterwards were significantly different (p less than 0.01), indicating an appreciable improvement in general health state, and at one year compared favourably with those from a normal male population. Analysis of responses to the study questionnaire showed that 65 of 89 patients (73%) were working at one year after operation with a further seven (8%) maintaining that they were fit to work but unable to find employment. The proportion of patients complaining of chest pain fell from 90% (88/98) before grafting to 19% (17/89) at one year after coronary artery bypass grafting, when 91% (81/89) patients maintained that their condition was either completely better or definitely improved. The significant positive factors affecting return to work and home activities were working before operation, short wait for operation, absence of breathlessness, and low physical mobility score in the Nottingham health profile (all p less than 0.001). CONCLUSIONS: Improvements were evident in general health state, symptoms, and activity at three months and one year after coronary artery bypass graft surgery. Interventions likely to influence outcomes included reduction in waiting times for operation; rehabilitation initiatives; and more attention to the quality of information given to patients, their relatives, and the community.

Activities of Daily Living↗
PubMed1991

The flavivirus envelope protein E: isolation of a soluble form from tick-borne encephalitis virus and its crystallization.

By the use of limited trypsin digestion of purified virions, we generated a membrane anchor-free and crystallizable form of the tick-borne encephalitis virus envelope glycoprotein E. It retained its reactivity with a panel of monoclonal antibodies, and only subtle structural differences from the native protein E were recognized. Treatment with the bifunctional cross-linker dimethylsuberimidate resulted in the formation of a dimer. Crystallization experiments yielded hexagonal rod-shaped crystals suitable for X-ray diffraction analysis.

Amino Acid Sequence↗
PubMed1990

Atomic structure of a fragment of human CD4 containing two immunoglobulin-like domains.

The structure of an N-terminal fragment of CD4 has been determined to 2.4 A resolution. It has two tightly abutting domains connected by a continuous beta strand. Both have the immunoglobulin fold, but domain 2 has a truncated beta barrel and a non-standard disulphide bond. The binding sites for monoclonal antibodies, class II major histocompatibility complex molecules, and human immunodeficiency virus gp120 can be mapped on the molecular surface.

Amino Acid Sequence↗
PubMed1990

Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA.

Protein-DNA recognition is often mediated by a small domain containing a recognizable structural motif, such as the helix-turn-helix or the zinc-finger. These motifs are compact structures that dock against the DNA double helix. Another DNA recognition motif, found in a highly conserved family of eukaryotic transcription factors including C/EPB, Fos, Jun and CREB, consists of a coiled-coil dimerization element the leucine-zipper and an adjoining basic region which mediates DNA binding. Here we describe circular dichroism and 1H-NMR spectroscopic studies of another family member, the yeast transcriptional activator GCN4. The 58-residue DNA-binding domain of GCN4, GCN4-p, exhibits a concentration-dependent alpha-helical transition, in accord with previous studies of the dimerization properties of an isolated leucine-zipper peptide. The GCN4-p dimer is approximately 70% helical at 25 degrees C, implying that the basic region adjacent to the leucine zipper is largely unstructured in the absence of DNA. Strikingly, addition of DNA containing a GCN4 binding site (AP-1 site) increases the alpha-helix content of GNC4-p to at least 95%. Thus, the basic region acquires substantial alpha-helical structure when it binds to DNA. A similar folding transition is observed on GCN4-p binding to the related ATF/CREB site, which contains an additional central base pair. The accommodation of DNA target sites of different lengths clearly requires some flexibility in the GCN4 binding domain, despite its high alpha-helix content. Our results indicate that the GCN4 basic region is significantly unfolded at 25 degrees C and that its folded, alpha-helical conformation is stabilized by binding to DNA.

Activating Transcription Factors↗
PubMed1990

Crystallization of the reovirus type 3 Dearing core. Crystal packing is determined by the lambda 2 protein.

Core particles of reovirus type 3 Dearing (T3D) crystallized in the face-centered cubic space group F432 with dimensions of 1270 A along each edge of the unit cell. Core particles of reovirus type 1 Lang (T1L) did not crystallize. Experiments with core particles derived from 27 different T1L x T3D reassortant viruses indicated that the L2 genome segment determined the capacity of cores to crystallize. This finding indicates important differences in the surface topography of the L2-translation product, the lambda 2 protein, of these two isolates, and suggests that important crystal contacts are mediated by this protein. These data are used to generate a model of the packing of reovirus core particles within the unit cell.

Crystallization↗