Structure of thermolysin refined at 1.6 A resolution.

Abstract The structure of the thermostable protease thermolysin has been refined by a restrained least-squares procedure at a nominal resolution of 1.6 A to a conventional R-value of 21.3% for 34,671 observed reflections (or R = 19.5% for reflections with F0 > 3σ(F0)). The refined structure was constrained to adhere to known stereochemistry, with root-mean-square deviations of 0.021 A from ideal bond lengths and 2.9 ° from ideal bond angles. The final model included 173 solvent molecules, which were given unit occupancies. Seven of these are “buried” within the protein. Atoms with the least apparent thermal motion tend to be those that are most deeply buried within the two domains of the structure. The active-site zinc is shown to have approximately tetrahedral co-ordination. Unusual features of the structure, confirmed by the refinement, include a cis-proline, a γ-turn, and a single turn of left-handed α-helix. The refinement shows that thermolysin does not contain unusual structures and supports our previous assertion that the thermostability of thermolysin and of thermostable proteins in general is due to a combination of factors which, in different instances, can include hydrophobic interactions, hydrogen bonding, ionic interactions, disulfide linkages, metal binding and other forms of stabilization.

[1]  Hans Frauenfelder,et al.  Temperature-dependent X-ray diffraction as a probe of protein structural dynamics , 1979, Nature.

[2]  B. Matthews,et al.  An oscillation data collection system for high‐resolution protein crystallography , 1981 .

[3]  D. Phillips,et al.  Crystallographic studies of the dynamic properties of lysozyme , 1979, Nature.

[4]  B. Matthews,et al.  The conformation of thermolysin. , 1974, The Journal of biological chemistry.

[5]  B. Matthews,et al.  Binding of hydroxamic acid inhibitors to crystalline thermolysin suggests a pentacoordinate zinc intermediate in catalysis. , 1982, Biochemistry.

[6]  W. Bode,et al.  The refined crystal structure of bovine beta-trypsin at 1.8 A resolution. II. Crystallographic refinement, calcium binding site, benzamidine binding site and active site at pH 7.0. , 1975, Journal of molecular biology.

[7]  M. Perutz,et al.  Stereochemical basis of heat stability in bacterial ferredoxins and in haemoglobin A2 , 1975, Nature.

[8]  B. Matthews,et al.  Molecular basis of thermostability in the lysozyme from bacteriophage T4 , 1979, Nature.

[9]  P Argos,et al.  Thermal stability and protein structure. , 1979, Biochemistry.

[10]  Michael G. Rossmann,et al.  Processing oscillation diffraction data for very large unit cells with an automatic convolution technique and profile fitting , 1979 .

[11]  J. Walker,et al.  Heat stability of a tetrameric enzyme, D-glyceraldehyde-3-phosphate dehydrogenase. , 1980, European journal of biochemistry.

[12]  J. Schellman,et al.  Mutations and protein stability , 1981, Biopolymers.

[13]  B. Matthews,et al.  The structure of thermolysin: an electron density map at 2-3 A resolution. , 1972, Journal of molecular biology.

[14]  B. Matthews,et al.  A crystallographic study of the complex of phosphoramidon with thermolysin. A model for the presumed catalytic transition state and for the binding of extended substances. , 1977, Journal of molecular biology.

[15]  V. Luzzati,et al.  Traitement statistique des erreurs dans la determination des structures cristallines , 1952 .

[16]  G J Williams,et al.  The Protein Data Bank: a computer-based archival file for macromolecular structures. , 1977, Journal of molecular biology.

[17]  L. Sieker,et al.  Water structure in a protein crystal: rubredoxin at 1.2 A resolution. , 1978, Journal of molecular biology.

[18]  J. C. Kendrew,et al.  The crystal structure of myoglobin: Phase determination to a resolution of 2 Å by the method of isomorphous replacement , 1961 .

[19]  F. Crick,et al.  The treatment of errors in the isomorphous replacement method , 1959 .

[20]  D. M. Blow,et al.  Structure of crystalline -chymotrypsin. V. The atomic structure of tosyl- -chymotrypsin at 2 A resolution. , 1972, Journal of molecular biology.

[21]  M. Sternberg,et al.  Dynamic information from protein crystallography. An analysis of temperature factors from refinement of the hen egg-white lysozyme structure. , 1979, Journal of molecular biology.

[22]  A. Fujishima,et al.  Electrochemical Photolysis of Water at a Semiconductor Electrode , 1972, Nature.

[23]  C. E. Klopfenstein,et al.  A computer controlled film scanner for X-ray crystallography , 1972 .

[24]  Kester Wr,et al.  Crystallographic study of the binding of dipeptide inhibitors to thermolysin: implications for the mechanism of catalysis. , 1977 .

[25]  L. Delbaere,et al.  Protein structure refinement: Streptomyces griseus serine protease A at 1.8 A resolution. , 1979, Journal of molecular biology.