Subsite binding in an RNase: structure of a barnase-tetranucleotide complex at 1.76-A resolution.

A set of subsites in barnase has been proposed from kinetic studies. A specific substrate analog, the tetradeoxynucleotide, CGAC, has been designed from this information. We report the crystal structure of its complex with barnase at 1.76-A resolution. The structure was solved by molecular replacement from a model of free barnase and refined to a crystallographic R factor of 19.0%. The stoichiometry of the asymmetric unit dimeric complex is [barnase:d(CGAC)]2, with 2-fold noncrystallographic symmetry. Each barnase molecule binds one oligonucleotide whereby the recognition site is occupied by guanine, and all three phosphate groups of the nucleotide make electrostatic interactions with basic residues in a strongly electropositive region at the bottom of the active site. The active-site His 102 packs against the adenine base of the nucleotide in an almost identical manner to the guanine base in the barnase-d(GpC) complex and defines a possible subsite in the Michaelis complex. The overall protein structure is unchanged on forming the complex with d(CGAC), but there are small differences in the active site and in crystal packing regions. The protein coordinates will be useful for theoretical calculations since some disorder induced by packing constraints in the crystals of the free enzyme are absent in the crystals of the complex. The interface of the dimer is formed by a His 102-adenine-adenine-His 102 face-to-face ring stack directly on the 2-fold axis. The edge of the adenine-adenine stack packs closely onto the face of a 3'-cytosine-3'-cytosine interaction, which has a "base-pair"-like conformation but too great a separation of the bases to form hydrogen bonds. This unusual arrangement is the major stabilizing interaction within the dimeric complex, since there are no direct protein-protein interactions. Using the structure of the complex as a starting point for model building, the nature of the enzyme-substrate and enzyme-transition state complexes is investigated.

[1]  A. Fersht,et al.  Crystal structural analysis of mutations in the hydrophobic cores of barnase. , 1994, Journal of molecular biology.

[2]  A. Fersht,et al.  Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy. , 1993, Biochemistry.

[3]  A. Lapthorn,et al.  Three‐dimensional structure of a barnase‐3'GMP complex at 2.2Å resolution , 1993, FEBS letters.

[4]  M. Guéron,et al.  A tetrameric DNA structure with protonated cytosine-cytosine base pairs , 1993, Nature.

[5]  A. Fersht,et al.  Barnase has subsites that give rise to large rate enhancements. , 1992, Biochemistry.

[6]  L Serrano,et al.  Effect of active site residues in barnase on activity and stability. , 1992, Journal of molecular biology.

[7]  W. Saenger,et al.  Crystal structure of ribonuclease T1 complexed with adenosine 2'-monophosphate at 1.8-A resolution. , 1993, The Journal of biological chemistry.

[8]  M Bycroft,et al.  Characterization of phosphate binding in the active site of barnase by site-directed mutagenesis and NMR. , 1991, Biochemistry.

[9]  U. Heinemann,et al.  Ribonuclease T1 with free recognition and catalytic site: crystal structure analysis at 1.5 A resolution. , 1991, Journal of molecular biology.

[10]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[11]  Sung-Hou Kim,et al.  Sparse matrix sampling: a screening method for crystallization of proteins , 1991 .

[12]  J. Janin,et al.  Crystal structure of a barnase-d(GpC) complex at 1.9 A resolution. , 1991, Journal of molecular biology.

[13]  F. Cordes,et al.  Evidence for a substrate-binding subsite in ribonuclease T1. Crystal structure of the complex with two guanosines, and model building of the complex with the substrate guanylyl-3',5'-guanosine. , 1991, The Journal of biological chemistry.

[14]  E J Dodson,et al.  Determination and restrained least-squares refinement of the structures of ribonuclease Sa and its complex with 3'-guanylic acid at 1.8 A resolution. , 1991, Acta crystallographica. Section B, Structural science.

[15]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[16]  A. Fersht,et al.  Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles. , 1990, Biochemistry.

[17]  K. Polyakov,et al.  Comparison of active sites of some microbial ribonucleases: structural basis for guanylic specificity. , 1990, Trends in biochemical sciences.

[18]  U. Heinemann,et al.  Crystal structure of guanosine-free ribonuclease T1, complexed with vanadate (V), suggests conformational change upon substrate binding. , 1989, Biochemistry.

[19]  A. Fersht,et al.  Kinetic characterization of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) and investigation of key residues in catalysis by site-directed mutagenesis. , 1989, Biochemistry.

[20]  J. Koepke,et al.  Three-dimensional structure of ribonuclease T1 complexed with guanylyl-2',5'-guanosine at 1.8 A resolution. , 1989, Journal of molecular biology.

[21]  J. Zelinka,et al.  Metabolism and Enzymology of Nucleic Acids: Including Gene Manipulations , 1989 .

[22]  A violent birth for Mercury , 1988, Nature.

[23]  A. McPherson,et al.  The mechanism of binding of a polynucleotide chain to pancreatic ribonuclease. , 1986, Science.

[24]  A. Fersht,et al.  Transition-state stabilization in the mechanism of tyrosyl-tRNA synthetase revealed by protein engineering. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Wolfram Saenger,et al.  Principles of Nucleic Acid Structure , 1983 .

[26]  C. Hill,et al.  The structural and sequence homology of a family of microbial ribonucleases , 1983 .

[27]  A. Wlodawer,et al.  Active site of RNase: neutron diffraction study of a complex with uridine vanadate, a transition-state analog. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[28]  M. J. Adams.  Preparation and Analysis of Protein Crystals , 1983 .

[29]  R. Porter.  Mobility and function in proteins and nucleic acids , 1983 .

[30]  U. Heinemann,et al.  Specific protein-nucleic acid recognition in ribonuclease T1–2′-guanylic acid complex: an X-ray study , 1982, Nature.

[31]  Alexander McPherson,et al.  Preparation and analysis of protein crystals , 1982 .

[32]  H R Drew,et al.  Structure of a B-DNA dodecamer. II. Influence of base sequence on helix structure. , 1981, Journal of molecular biology.

[33]  F. Eckstein,et al.  Absolute Stereochemistry of the Second Step of Ribonuclease Action , 1970, Nature.

[34]  A. Wilson,et al.  The probability distribution of X-ray intensities , 1949 .