Ribonuclease crystal structure

Leucine residues 2, 5, 7, 12, 20, and 24 of the motif are invariant in both type A and type B repeats of the ribonuclease inhibitor. An examination of more than 500 tandem repeats from 68 different proteins has shown that residues 20 and 24 can be other hydrophobic residues, whereas the remaining four leucine residues are present in all repeats. On the basis of the crystal structure of the ribonuclease inhibitor and the important structural role of these leucine residues, it has been possible to construct plausible structural models of several other proteins with leucine-rich motifs, such as the extracellular domains of the thyrotropin and gonadotropin receptors. 

Kobe, B., Deisenhofer, J. Crystal structure of porcine ribonuclease inhibitor, a protein with leucine-rich repeats. Nature 366 751-756, 1993. 

Wlodawer A, Bott R, Sjolin L. The refined crystal structure of ribonuclease A at 2.0 A resolution. I. Biol. Chem. 1982 257 1325-1332. 

S. Capasso, A. Di Donato, L. Esposito, F. Sica, G. Sorrentino, L. Vitagliano, A. Zagari, L. Mazzarella, Deamidation in Proteins The Crystal Structure of Bovine Pancreatic Ribonuclease with an Isoaspartyl Residue at Position 67 , J. Mol. Biol. 1996, 257, 492 -496. 

Despite considerable biochemical work, high-resolution crystal structure determination of native RNase A and S, and some medium-resolution studies of RNase A-inhibitor complexes, a number of questions existed concerning the details of the catalytic mechanism and the role of specific amino acids. Study of the low-temperature kinetics and three-dimensional structures of the significant steps of the ribonuclease reaction was designed to address the following questions. 

Davies JF, Hostomska Z, Hostomsky Z, Jordan SR, Matthews DA. Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. Science 1991 252 88-95. 

The enzyme consists of a single polypeptide chain of Mr 13 680 and 124 amino acid residues.187,188 The bond between Ala-20 and Ser-21 may be cleaved by subtilisin. Interestingly, the peptide remains attached to the rest of the protein by noncovalent bonds. The modified protein, called ribonuclease S, and the native protein, now termed ribonuclease A, have identical catalytic activities. Because of its small size, its availability, and its ruggedness, ribonuclease is very amenable to physical and chemical study. It was the first enzyme to be sequenced.187 The crystal structures of both forms of the enzyme were solved at 2.0-A resolution several years ago.189,190 Subsequently, crystal structures of many complexes of the enzyme with substrate and transition analogues and products have been solved at very high resolution.191 Further, because the catalytic activity depends on the ionizations of two histidine residues, the enzyme has been extensively studied by NMR (the imidazole rings of histidines are easily studied by this method—see Chapter 5). 

Fig. 9. The conformation adopted by a leucine-rich repeat (LRR) is that of a /9-strand followed by an o-helix. In porcine ribonuclease inhibitor, a /9-strand (residues 2-8) is connected to an o-helix (residues 14—27) by a connecting loop (residues 9-13). A horseshoe-shaped structure is formed and is exemplified in the crystal structure of ribonuclease inhibitor (PDB 1A4Y Kobe and Deisenhofer, 1993). This has an inner concave surface formed by curved /9-sheets and an outer convex surface formed by oh el ices. The leucines and other large apolar residues form the hydrophobic core of the structure.

Hambley, T.W., R.J. Judd, and P.A. Lay. 1992. Synthesis and crystal structure of a vanadium(V) complex with a 2-hydroxy acid ligand A structural model of both vanadium(V) transferrin and ribonuclease complexes with inhibitors. Inorg. Chem. 31 343-345. 

Kostrewa, D., H.W. Choe, U. Heinemann, and W. Saenger. 1989. Crystal structure of guanosine-free ribonuclease T1, complexed with vanadate(V), suggests conformation change upon substrate binding. Biochemistry 28 7592-7600. 

Two other types of specific side-chain interactions have been proposed to stabilize the a helix formed by C-peptide (Shoemaker et al., 1987b). A salt bridge between Glu-2 and Arg-10 has been detected in the crystal structure of ribonuclease A (Wlodawer and Sjolin, 1983) as well as in C-peptide in aqueous solution (Rico et al., 1986). This salt bridge also fixes the N-terminal boundary of the helix between Glu-2 and Thr-3. It is not sterically possible to make this ion pair if Glu-2 is part of the helix. Synthetic studies in which either Glu-2 or Arg-10 is replaced by Ala have provided support for the importance of this interaction in stabilizing the a-... 

Ribonuclease S, of knowm crystal structure (10), presents an instructive case. Except in the region of residues 18-23, it is very similar to RNase A (11). CjNj modifies known salt-bridge pairs (5). The distance between the C s of ALA20 and SER 21 is 27 A calculated from the X-ray diffraction based coordinates (12), however, considerable uncertainty exists relative to structural parameters for residues 18-23 (12). Are residues 20 and 21 transiently associated and susceptible to C2N2 driven condensation The answer appears to be yes but not as a reestablished peptide bond. Instead, it appears to be a depsi-pepMe ester link. 

The presence of the glycan (light blue) on ribonuclease B reduces the amide proton/deuteiium exchange rates compared with ribonuclease A for extensive regions of the peptide backbone (shown in red) both local to and remote from the glycosylation site [47]. The glycoprotein structure is based on the crystal structure of ribonuclease B [53] and one of the structures of MangGlcNAc2 determined by NMR and molecular dynamics. (Reprinted from [44] with permission from Elsevier), [54]... 

Nonaka, T., Nakamura, K. T., Uesugi, S., et al. (1993) Crystal structure of ribonuclease Ms (as a ribonuclease T1 homolog) complexed with a guanylyl-3, 5 -cytidine analog. Biochemistry, 32, 11825-11837. 

The crystal structure of both ribonuclease A and S have been determined and residues 2-13 form two-three turns of a-helix (4,). In the dissociated state, S-peptide contains little detectable helix either by circular dichroism (5) or by NMR (6,7). Either the S-protein induces the bound conformation during the association or a conformer too limited in concentration to be detected by spectroscopy is preferentially bound. It has been argued (3) on kinetic grounds that the induced-fit or "zipper" hypothesis is more likely, but no relevant experimental data exists on this or other systems to our knowledge. 

The first protein crystal structure, myoglobin, was solved in 1960. The second, lysozyme, followed in 1965. In 1967 three structures were solved ribonuclease, chymotrypsin and carboxypeptidase. Thereafter, the number solved has increased almost exponentially year by year so that by 1979 there were some 161 structures known, at least at the level of tracing the fold of the polypeptide chain [6]. To date, there are well over 200 structures solved, but this number includes several structures of the same protein in a different crystal form or from a different species. Some protein structures are illustrated in Figure 1. 

Comparative studies on the same protein crystallised under different conditions and in different space groups show that the structures are essentially the same. For example, monoclinic (C2) subtilisin crystallised from 2.1 M ammonium sulphate, pH 5.9, has the same crystal structure as monoclinic (P2 J subtilisin crystallised from a 55% acetone/water mixture at pH 9.1 [152]. Other examples include tetragonal and triclinic lysozyme, orthorhombic and trigonal trypsin, and trigonal and monoclinic ribonuclease. 

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