Determination and analysis of the 2 A-structure of copper, zinc superoxide dismutase.
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Binding Sites↗
Biomedical subjects
E D Getzoff
Publications and source records attributed to E D Getzoff.
The beta bulge: a common small unit of nonrepetitive protein structure.
A beta bulge is a region between two consecutive beta-type hydrogen bonds which includes two residues (positions 1 and 2) on one strand opposite a single residue (position x) on the other strand. Compared to regular beta structure, a beta bulge puts the usual alternation of side-chain direction out of register on one of the strands, introduces a slight bend in the beta sheet, and locally accentuates the usual right-handed strand twist. Almost all beta bulges are between antiparallel strands, usually between a narrow rather than a wide pair of hydrogen bonds. Ninety-one examples are listed. The two commonest types are the "classic" beta bulge, with position 1 in approximately alpha-helical conformation, and the "G1" beta bulge, with a required glycine at position 1 in approximately left-handed alpha-helical conformation, G1 bulges almost always occur in combination with a type II tight turn. The functional roles of beta bulges probably include compensating for the effects of a single-residue insertion or deletion within beta structure and providing the strong local twist required for form closed beta barrel structures.
Amino Acid Sequence↗
Unraveling the effect of changes in conformation and compactness at the antibody V(L)-V(H) interface upon antigen binding.
We have analyzed conformational changes that occur at the interface between the light (V(L)) and heavy (V(H)) chains in antibody variable fragments upon binding to antigens. We wrote and applied the Tiny Probe program that computes the buried atomic contact surface area of three-dimensional structures to evaluate changes in compactness of the V(L)-V(H) interface between bound and unbound antibodies. We found three categories of these changes, which correlated with the size of the antigen. Upon binding, medium-sized nonprotein antigens cause an opening of the V(L)-V(H) interface (less compact), small antigens or haptens cause a closure of the interface (more compact), whereas large protein antigens have little effect on the compactness of the V(L)-V(H) interface. The largest changes in the atomic buried contact surface area at the V(L)-V(H) interface occur in residue pairs providing two 'shock absorbers' between the edge beta-strands of the V(L) and V(H) beta-sheets forming the antibody binding site. Importantly, the correlation between the size of antigens and conformational changes indicates that the V(L)-V(H) interface in antibodies plays a significant role in the antigen binding process. Furthermore, as the energy involved in such a motion is significant (up to 3 kcal/mol), these results provide a general mechanism for how residues distant from the combining site can significantly alter the affinity of an antibody for its antigen. Thus, mutations introduced at the V(L)-V(H) interface can be used to change antibody binding affinity with antigens. Due to the tightly packed V(L)-V(H) interface, the introduction of random mutations is not advisable. Rather our analysis suggests that concerted mutations of residues preceding CDRL2 and following CDRH3 or residues preceding CDRH2 and at the end of CDRL3 are most likely to alter or improve antigen binding affinity.
Antigen-Antibody Reactions↗
Structure and mechanism of copper, zinc superoxide dismutase.
Copper, zinc superoxide dismutase (SOD) catalyses the very rapid two-step dismutation of the toxic superoxide radical (O-2) to molecular oxygen and hydrogen peroxide through the alternate reduction and oxidation of the active-site copper. We report here that after refitting and further refinement of the previous 2 A structure of SOD2, analysis of the new model and its calculated molecular surface shows an extensive surface topography of sequence-conserved residues stabilized by underlying tight packing and H-bonding. There is a single, highly complementary position for O-2 to bind to both the Cu(II) and activity-important Arg 141 with correct geometry; two water molecules form a ghost of the superoxide in this position. The geometry and molecular surface of the active site, together with biochemical data, suggest a specific model for the enzyme mechanism.
Binding Sites↗
Electrostatic recognition between superoxide and copper, zinc superoxide dismutase.
Electrostatic forces have been implicated in a variety of biologically important molecular interactions including drug orientation by DNA, protein folding and assembly, substrate binding and catalysis and macromolecular complementarity with inhibitors, drugs and hormones. To examine enzyme-substrate interactions in copper, zinc superoxide dismutase (SOD), we developed a method for the visualization and analysis of an enzyme's three-dimensional electrostatic vector field that allows the contributions of specific residues to be identified. We report here that the arrangement of electrostatic charges in SOD promotes productive enzyme-substrate interaction through substrate guidance and charge complementarity: sequence-conserved residues create an extensive electrostatic field that directs the negatively charged superoxide (O-2) substrate to the highly positive catalytic binding site at the bottom of the active-site channel. Dissection of the electrostatic potential gradient indicated the relative contributions of individual charged residues: Lys 134 and Glu 131 seem to have important roles in directing the long-range approach of O-2, while Arg 141 has local orienting effects. The reported methods of analysis may have general application for the elucidation of intermolecular recognition processes.
Binding Sites↗
The reactivity of anti-peptide antibodies is a function of the atomic mobility of sites in a protein.
To study the nature of antigenic recognition, antibodies have been prepared against a set of peptide sequences representing both highly mobile and well-ordered regions of myohaemerythrin, based on X-ray crystallographic temperature factors. Anti-peptide antibodies against highly mobile regions react strongly with the native protein; anti-peptide antibodies from well-ordered regions do not. Mobility is a major factor in the recognition of the native protein by anti-peptide antibodies; this may be of general significance in protein-protein interactions.
Animals↗
Understanding the structure and antigenicity of gonococcal pili.
Pili--filamentous protein structures found on the cell surface of many infectious gram-negative bacteria--often are virulence factors that mediate adherence to host epithelial cells. The pilus, a major surface antigen of the gonococcus, is formed by the specific association of thousands of repeating identical protein subunits (pilin). Structural studies of the pilin protein and the pilus fiber may aid the rational design of a peptide-based vaccine by providing information on the antigenic surface of pili that includes the identification of sequence-distant antigenic regions that are localized to single areas of the three-dimensional structure. Preliminary results suggest that the pilin subunit has structural similarity to the 4-alpha-helix bundle fold in proteins such as the coat protein subunit of tobacco mosaic virus and myohemerythrin. Therefore, the monomeric protein myohemeythrin was used to identify structural correlations for antigenic determinants on 4-alpha-helix bundle proteins such as pilin.
Antigens, Bacterial↗