The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.

Since its first isolation, bovine beta-lactoglobulin (BLG) has been an enigma: although it is abundant in the whey fraction of milk, its function is still not clear. The results of the many physicochemical studies on the protein need a structural interpretation. We report here the structure of the orthorhombic crystal form of cow BLG at pH 7.6, at a resolution of 2.8 A. It has an unusual protein fold, composed of two slabs of antiparallel beta-sheet, which shows a remarkable similarity to plasma retinol-binding protein. A possible binding site for retinol in BLG has been identified by model-building. This suggests a role for BLG in vitamin A transport and we have discovered specific receptors for the BLG-retinol complex in the intestine of neonate calves.

The conserved, abundant chromosomal protein HMG1 consists of two highly homologous, folded, basic DNA‐binding domains, each of approximately 80 amino acid residues, and an acidic C‐terminal tail. Each folded domain represents an ‘HMG box’, a sequence motif recently recognized in certain sequence‐specific DNA‐binding proteins and which also occurs in abundant HMG1‐like proteins that bind to DNA without sequence specificity. The HMG box is defined by a set of highly conserved residues (most distinctively aromatic and basic) and appears to define a novel DNA‐binding structural motif. We have expressed the HMG box region of the B‐domain of rat HMG1 (residues 88–164 of the intact protein) in Escherichia coli and we describe here the determination of its structure by 2D 1H‐NMR spectroscopy. There are three alpha‐helices (residues 13–29, 34–48 and 50–74), which together account for approximately 75% of the total residues and contain many of the conserved basic and aromatic residues. Strikingly, the molecule is L‐shaped, the angle of approximately 80 degrees between the two arms being defined by a cluster of conserved, predominantly aromatic, residues. The distinctive shape of the HMG box motif, which is distinct from hitherto characterized DNA‐binding motifs, may be significant in relation to its recognition of four‐way DNA junctions.

A high-resolution solution structure of the GDP form of a truncated version of the ras p21 protein (residues 1-166) has been determined using NMR spectroscopy. Ras p21 is the product of the human ras protooncogene and a member of a ubiquitous eukaryotic gene family which is highly conserved in evolution. A virtually complete assignment (13C, 15N, and 1H), including stereospecific assignments of 54 C beta methylene protons and 10 C gamma methyl protons of valine residues, was obtained by analysis of three- and four-dimensional (3D and 4D) heteronuclear NMR spectra using a newly developed 3D/4D version of the ANSIG software. A total of 40 converged structures were computed from 3369 experimental restraints consisting of 3,167 nuclear Overhauser effect (NOE) derived distances, 14 phi and 54 chi 1 torsion angle restraints, 109 hydrogen bond distance restraints, and an additional 25 restraints derived from literature data defining interactions between the GDP ligand, the magnesium ion, and the protein. The structure in the region of residues 58-66 (loop L4), and to a lesser degree residues 30-38 (loop L2), is ill-defined. Analysis of the dynamics of the backbone 15N nuclei in the protein showed that residues within the regions 58-66, 107-109, and, to a lesser degree, 30-38 are dynamically mobile on the nanosecond time scale. The root mean square (rms) deviations between the 40 solution structures and the mean atomic coordinates are 0.78 A for the backbone heavy atoms and 1.29 A for all non-hydrogen atoms if all residues (1-166) are included in the analysis. If residues 30-38 and residues 58-66 are excluded from the analysis, the rms deviations are reduced to 0.55 and 1.00 A, respectively. The structure was compared to the most highly refined X-ray crystal structure of ras p21.GDP (1-189) [Milburn, M. V., Tong, L., de Vos, A. M., Brünger, A. T., Yamaizumi, Z., Nishimura, S., & Kim, S.-H. (1990) Science 24, 939-945]. The structures are very similar except in the regions found to be mobile by NMR spectroscopy. In addition, the second alpha-helix (helix-2) has a slightly different orientation. The rms deviation between the average of the solution structures and the X-ray crystal structure is 0.94 A for the backbone heavy atoms if residues 31-37 and residues 59-73 are excluded from the analysis.

The solution conformation of the antibacterial polypeptide cecropin A from the Cecropia moth is investigated by nuclear magnetic resonance (NMR) spectroscopy under conditions where it adopts a fully ordered structure, as judged by previous circular dichroism studies [Steiner, H. (1982) FEBS Lett. 137, 283-287], namely, 15% (v/v) hexafluoroisopropyl alcohol. By use of a combination of two-dimensional NMR techniques the 1H NMR spectrum of cecropin A is completely assigned. A set of 243 approximate interproton distance restraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 32 distance restraints for the 16 intrahelical hydrogen bonds identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing [Nilges, M., Clore, G.M., & Gronenborn, A.M. (1988) FEBS Lett. 229, 317-324]. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Seven independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 21 calculated structures satisfy the experimental restraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 21 converged structure indicates that there are two helical regions extending from residues 5 to 21 and from residues 24 to 37 which are very well defined in terms of both atomic root mean square differences and backbone torsion angles. For the two helical regions individually the average backbone rms difference between all pairs of structures is approximately 1 A. The long axes of the two helices lie in two planes, which are at an angle of 70-100 degrees to each other. The orientation of the helices within these planes, however, cannot be determined due to the paucity of NOEs between the two helices.

No abstract

The function of the cellulose-binding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site-directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain. The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose. Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge-shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD. However, there was no effect on the activity toward small oligosaccharide (4-methylumbelliferyl beta-D-lactoside). The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important. However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants. The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI. These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose.

We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.In the complex molecular interplay between a pathogen and its human host, protein-protein interactions play important roles. For instance, bacteria express surface proteins that interact with abundant human extracellular proteins with high affinity and specificity. In human plasma, albumin (HSA) 1 and immunoglobulins (Ig) are the quantitatively dominating proteins, and significant human pathogens have developed surface proteins that bind these and other plasma proteins (for references, see Ref 1). Two of the most well known such proteins are protein A of Staphylococcus aureus (2) and protein G of group C and G streptococci (3, 4), which both bind to the Fc region of IgG. In 1980, Myhre and Kronvall (5) reported that HSA could bind to the surface of various streptococcal species, including group C and G streptococci. It was later found that protein G was responsible also for the interaction with HSA (6). Protein G has separate binding domains for IgG and HSA (7,8), and as a result C and G streptococci are in vivo covered with an inner layer of IgG and an outer layer of HSA. Peptostreptococcus magnus are strictly anaerobic bacteria that are part of the indigenous human flora of the skin, oral cavity, and gastrointestinal and urogenital tracts. Some isolates of this species bind HSA (9), and notably these isolates are mostly from patients with deep wound infections (10), suggesting that HSA binding turns the commensal P. magnus into a potential pathogen. The surface protein of P. magnus binding HSA is called PAB, and it contains a domain of 45 residues showing a high degree of sequence homology with the HSA binding domains of protein G (11). Analysis of the gene encoding PAB suggested that this domain originates from protein G and has been transferred to and introduced into the pab gene through the action of a conjugational plasmid (related to pCF10 of Enterococcus faecalis) followed by a recombinational event (11). This interspecies exchange of a structurally well defined motif repre...