Crystal Structure of LexA A Conformational Switch for Regulation of Self-Cleavage

LexA repressor undergoes a self-cleavage reaction. In vivo, this reaction requires an activated form of RecA, but it occurs spontaneously in vitro at high pH. Accordingly, LexA must both allow self-cleavage and yet prevent this reaction in the absence of a stimulus. We have solved the crystal structures of several mutant forms of LexA. Strikingly, two distinct conformations are observed, one compatible with cleavage, and the other in which the cleavage site is approximately 20 A from the catalytic center. Our analysis provides insight into the structural and energetic features that modulate the interconversion between these two forms and hence the rate of the self-cleavage reaction. We suggest RecA activates the self-cleavage of LexA and related proteins through selective stabilization of the cleavable conformation.

[1]  J. A. Rupley,et al.  In vitro analysis of mutant LexA proteins with an increased rate of specific cleavage. , 1992, Journal of molecular biology.

[2]  G. Wagner,et al.  Dimerization of the UmuD' protein in solution and its implications for regulation of SOS mutagenesis , 1997, Nature Structural Biology.

[3]  M. Qasim,et al.  What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? , 2000, Biochimica et biophysica acta.

[4]  B. Delmas,et al.  Role of Ser-652 and Lys-692 in the protease activity of infectious bursal disease virus VP4 and identification of its substrate cleavage sites. , 2000, The Journal of general virology.

[5]  J. W. Little,et al.  Autodigestion of lexA and phage lambda repressors. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[6]  M. Daune,et al.  Large-scale purification, oligomerization equilibria, and specific interaction of the LexA repressor of Escherichia coli. , 1985, Biochemistry.

[7]  G J Barton,et al.  ALSCRIPT: a tool to format multiple sequence alignments. , 1993, Protein engineering.

[8]  B. Honig,et al.  Classical electrostatics in biology and chemistry. , 1995, Science.

[9]  J. Kraut.  Serine proteases: structure and mechanism of catalysis. , 1977, Annual review of biochemistry.

[10]  M. Paetzel,et al.  Crystal structure of a bacterial signal peptidase in complex with a β-lactam inhibitor , 1998, Nature.

[11]  M. Daune,et al.  In vitro binding of LexA repressor to DNA: evidence for the involvement of the amino‐terminal domain. , 1986, The EMBO journal.

[12]  M. Lewis,et al.  Crystal Structure of the λ Repressor C-Terminal Domain Provides a Model for Cooperative Operator Binding , 2000, Cell.

[13]  D. Lilley,et al.  DNA Repair , 1998, Nucleic Acids and Molecular Biology.

[14]  M. Ptashne.  A Genetic Switch , 1986 .

[15]  J. W. Little,et al.  Isolation and characterization of noncleavable (Ind-) mutants of the LexA repressor of Escherichia coli K-12 , 1988, Journal of bacteriology.

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

[17]  K. Roland,et al.  Reaction of LexA repressor with diisopropyl fluorophosphate. A test of the serine protease model. , 1990, The Journal of biological chemistry.

[18]  K. Roland,et al.  Cleavage of LexA repressor. , 1994, Methods in enzymology.

[19]  J. W. Little.  Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease. , 1991, Biochimie.

[20]  D. Mount,et al.  The SOS regulatory system of Escherichia coli , 1982, Cell.

[21]  E. G. Frank,et al.  The UmuD' protein filament and its potential role in damage induced mutagenesis. , 1996, Structure.

[22]  J. W. Little,et al.  Autodigestion and RecA-dependent cleavage of Ind- mutant LexA proteins. , 1989, Journal of molecular biology.

[23]  J. W. Little,et al.  Mutant LexA proteins with specific defects in autodigestion. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Thornton,et al.  Identification, classification, and analysis of beta‐bulges in proteins , 1993, Protein science : a publication of the Protein Society.

[25]  J. Roberts,et al.  Escherichia coli recA gene product inactivates phage lambda repressor. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[27]  B. Matthews,et al.  Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme. , 1991, Biochemistry.

[28]  B. Cravatt,et al.  Chemical and mutagenic investigations of fatty acid amide hydrolase: evidence for a family of serine hydrolases with distinct catalytic properties. , 1999, Biochemistry.

[29]  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.

[30]  E. G. Frank,et al.  Intermolecular cleavage by UmuD-like mutagenesis proteins. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. Woodgate,et al.  Intermolecular cleavage by UmuD-like enzymes: identification of residues required for cleavage and substrate specificity. , 1999, Journal of molecular biology.

[32]  R. Kaptein,et al.  Solution structure of the LexA repressor DNA binding domain determined by 1H NMR spectroscopy. , 1994, The EMBO journal.

[33]  G. Walker,et al.  A model for a umuDC-dependent prokaryotic DNA damage checkpoint. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. A. Dougherty,et al.  Cation-π interactions in structural biology , 1999 .

[35]  B. Diner,et al.  Crystal structures of the photosystem II D1 C-terminal processing protease , 2000, Nature Structural Biology.

[36]  R. Sauer,et al.  Lambda repressor inactivation: properties of purified ind- proteins in the autodigestion and RecA-mediated cleavage reactions. , 1986, Journal of molecular biology.

[37]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[38]  E. G. Frank,et al.  Structure of the UmuD′ protein and its regulation in response to DNA damage , 1996, Nature.

[39]  J. W. Little,et al.  LexA and λ Cl repressors as enzymes: Specific cleavage in an intermolecular reaction , 1993, Cell.

[40]  R. Scheuermann,et al.  UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[41]  J. W. Little.  LexA cleavage and other self-processing reactions , 1993, Journal of bacteriology.

[42]  D. Bacon,et al.  A fast algorithm for rendering space-filling molecule pictures , 1988 .

[43]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[44]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[45]  V S Lamzin,et al.  Automated refinement for protein crystallography. , 1997, Methods in enzymology.

[46]  R. Sauer,et al.  P22 c2 repressor. Domain structure and function. , 1983, Journal of Biological Chemistry.

[47]  D. Mount,et al.  Cleavage of the Escherichia coli lexA protein by the recA protease. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[48]  D E McRee,et al.  XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. , 1999, Journal of structural biology.

[49]  J. W. Little,et al.  Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[50]  D E Wemmer,et al.  Two-state allosteric behavior in a single-domain signaling protein. , 2001, Science.

[51]  S. N. Slilaty,et al.  The role of electrostatic interactions in the mechanism of peptide bond hydrolysis by a Ser-Lys catalytic dyad. , 1991, Protein engineering.

[52]  M. Paetzel,et al.  Correction: Crystal structure of a bacterial signal peptidase in complex with a β-lactam inhibitor , 1998, Nature.