NORMAL GENOMIC

• Andrade MA, Perez-Iratxeta C, Ponting CP
• Protein repeats: structures, functions, and evolution.
• J Struct Biol. 2001; 134: 117-31
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• Internal repetition within proteins has been a successful strategem on multiple separate occasions throughout evolution. Such protein repeats possess regular secondary structures and form multirepeat assemblies in three dimensions of diverse sizes and functions. In general, however, internal repetition affords a protein enhanced evolutionary prospects due to an enlargement of its available binding surface area. Constraints on sequence conservation appear to be relatively lax, due to binding functions ensuing from multiple, rather than, single repeats. Considerable sequence divergence as well as the short lengths of sequence repeats mean that repeat detection can be a particularly arduous task. We also consider the conundrum of how multiple repeats, which show strong structural and functional interdependencies, ever evolved from a single repeat ancestor. In this review, we illustrate each of these points by referring to six prolific repeat types (repeats in beta-propellers and beta-trefoils and tetratricopeptide, ankyrin, armadillo/HEAT, and leucine-rich repeats) and in other less-prolific but nonetheless interesting repeats. Copyright 2001 Academic Press.

• Evdokimov AG, Anderson DE, Routzahn KM, Waugh DS
• Unusual Molecular Architecture of the Yersinia pestis Cytotoxin YopM: A Leucine-rich Repeat Protein with the Shortest Repeating Unit.
• J Mol Biol. 2001; 312: 807-21
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• Many Gram-negative bacterial pathogens employ a contact-dependent (type III) secretion system to deliver effector proteins into the cytosol of animal or plant cells. Collectively, these effectors enable the bacteria to evade the immune response of the infected organism by modulating host-cell functions. YopM, a member of the leucine-rich repeat protein superfamily, is an effector produced by the bubonic plague bacterium, Yersinia pestis, that is essential for virulence. Here, we report crystal structures of YopM at 2.4 and 2.1 A resolution. Among all leucine-rich repeat family members whose atomic coordinates have been reported, the repeating unit of YopM has the least canonical secondary structure. In both crystals, four YopM monomers form a hollow cylinder with an inner diameter of 35 A. The domain that targets YopM for translocation into eukaryotic cells adopts a well-ordered, alpha-helical conformation that packs tightly against the proximal leucine-rich repeat module. A similar alpha-helical domain can be identified in virulence-associated leucine-rich repeat proteins produced by Salmonella typhimurium and Shigella flexneri, and in the conceptual translation products of several open reading frames in Y. pestis.

• Kobe B, Deisenhofer J
• Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A.
• J Mol Biol. 1996; 264: 1028-43
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• We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an Rcryst of 19.4% at 2.5 A resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the p1 phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 A2 of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (Ki = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse ribonucleases. However, the inhibition is lost with amphibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer.

• Shapiro R, Riordan JF, Vallee BL
• LRRning the RIte of springs.
• Nat Struct Biol. 1995; 2: 350-4
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• The determination of the crystal structure of the ribonuclease inhibitor-ribonuclease A complex provides exciting new insight on how the leucine-rich repeat allows a single molecule to get around the problem of inhibiting an entire family of enzymes.

• Neumann U, Hofsteenge J, Arkema AH, Dijkstra BW
• Crystallization of porcine liver ribonuclease inhibitor a member of the family of proteins containing leucine-rich repeats.
• J Mol Biol. 1993; 231: 505-8
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• Single crystals of the RNase inhibitor from porcine liver have been obtained from 30 to 34% saturated ammonium sulphate solutions at pH 6.0 to 7.2, containing 20 mM dithiothreitol, at room temperature over a period of two to three weeks. Because the inhibitor contains 30 1/2-cystinyl residues, all of which occur in the free thiol form, crystallization experiments were carried out in a desiccator under a nitrogen atmosphere. The crystals belong to the tetragonal space group I4, with cell dimensions a = b = 134.76 A and c = 83.65 A. The asymmetric part of the unit cell contains two molecules with a molecular mass of 49,093 Da, as could be shown with a self-rotation function calculated in the resolution range 10.0 to 3.2 A. The crystals diffract to at least 3.2 A resolution and are suitable for an X-ray structure determination.

• Lee FS, Vallee BL
• Modular mutagenesis of human placental ribonuclease inhibitor, a protein with leucine-rich repeats.
• Proc Natl Acad Sci U S A. 1990; 87: 1879-83
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• Human placental ribonuclease inhibitor (PRI) is a potent protein inhibitor of pancreatic ribonucleases and the homologous blood vessel-inducing protein angiogenin. Although inhibition by PRI occurs with a 1:1 stoichiometry, its primary structure is composed predominantly of seven internal leucine-rich repeats. These internal repeats were systematically deleted either singly or in combination by "modular" mutagenesis. Deletion of repeat units 3 plus 4 or repeat unit 6 results in mutants that both bind to and inhibit ribonuclease A. Therefore, the angiogenin/ribonuclease binding site in PRI must reside primarily or entirely in repeats 1, 2, 5, or 7, the short N- or C-terminal segments, or a combination of these. Deletion of repeat units 3-5, 5-6, or 5 alone results in mutants that exhibit only binding activity. Hence, the binding site cannot reside exclusively in repeat 5. Other internal deletions or N- or C-terminal deletions of 6-86% of the protein all abolish activity. These results suggest that PRI has a modular structure, with one primary structural repeat constituting one module. The approach taken may be applicable to other proteins with repeat structures.