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Nanoviruses are a family of plant viruses that possess a genome of multiple circular single-stranded DNA (ssDNA) components and are strikingly similar in their replication mode to the plant geminiviruses and to the circoviruses that infect birds or mammals. These viruses multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyze the initiation of replication on a double-stranded DNA (dsDNA) intermediate and the resolution of the ssDNA into circles. Here we report the solution NMR three-dimensional structure of the endonuclease domain from the master Rep (M-Rep) protein of faba bean necrotic yellows virus (FBNYV), a representative of the nanoviruses. The domain comprises amino acids 2-95 (M-Rep2-95), and its global fold is similar to those previously described for the gemini- and circovirus Rep endonuclease domains, consisting of a central 5-stranded antiparallel beta-sheet covered on one side by an alpha-helix and irregular loops and on the other, more open side of the domain, by an alpha-helix containing the catalytic tyrosine residue (the catalytic helix). Longer domain constructs extending to amino acids 117 and 124 were also characterized. They contain an additional alpha-helix, are monomeric, and exhibit catalytic activity indistinguishable from that of M-Rep2-95. The binding site for the catalytic metal was identified by paramagnetic broadening and maps to residues on the exposed face of the central beta-sheet. A comparison with the previously determined Rep endonuclease domain structures of tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus, and that of porcine circovirus type 2 (PCV2) Rep allows the identification of a positively charged surface that is most likely involved in dsDNA binding, and reveals common features shared by all endonuclease domains of nanovirus, geminivirus, and circovirus Rep proteins.

Alpha-sarcin is an exquisitely specific ribonuclease that binds and cleaves a single phosphodiester bond in the large rRNA of the eukaryotic ribosome, inactivating it. To better understand this remarkable activity, the contributions of the active site residues (His 50, Glu 96, and His 137) to the conformational stability have been determined as a function of pH using variant proteins containing uncharged substitutes. Wild-type alpha-sarcin and the variants are maximally stable near pH 5.5, coinciding with the pH of optimal activity. A comparison of the stability vs pH profiles determined by thermal denaturation experiments to those calculated on the basis of pKa values shows that the charged forms of Glu 96 and His 137 compromise the enzyme's stability, lowering it. In contrast to barnase, there is little evidence for significant electrostatic interactions in the denatured states of alpha-sarcin or its active site variants between pH 3.5 and pH 8.5. Alpha-sarcin contains a long beta-hairpin and surface loops which are highly positively charged and which play key roles in membrane translocation and in ribosome binding. These positive charges decrease the stability of alpha-sarcin, particularly below pH 5. Hydrogen exchange measurements have been performed at pH 5.5 and reveal that the catalytic residues are firmly anchored in highly stable elements of secondary structure. Significant, though lower, levels of protection are observed for many amide protons in the positively charged beta-hairpin and long loops.

Nuclear magnetic resonance (NMR) (15)N relaxation methods have been used to characterize the backbone dynamics of the N-terminal core domain of the HIV-1 capsid protein (CA(151)). The domain, which has an unusually flat, triangular shape, tumbles in solution at 28 degrees C with an effective rotational correlation time of 11.5 ns. Relaxation data for backbone amides in the domain's seven alpha-helices are indicative of fully anisotropic rotational diffusion. The principal axes of the rotational diffusion tensor calculated from the NMR data are aligned to within 12-23 degrees of the principal axes of the inertial tensor, with the axis of fastest rotational diffusion coincident with that of minimal inertia, and vice versa. Large variations in the (15)N-(1)H nuclear Overhauser effects for individual amino acids correlate with the degree of convergence in the previously calculated NMR structure. In particular, the partially disordered residues Val86-Arg97 that contain the human cyclophilin A (CypA) packaging signal have (15)N heteronuclear NOEs and transversal relaxation rates consistent with a high degree of dynamic conformational averaging. The N-terminal domain of a CA mutant (G94D) that confers both resistance to and dependence on cyclosporin A analogues was also analyzed. Our results indicate that this mutation does not influence the conformation or dynamics of CA(151), and therefore probably affects the function of the protein by modifying essential intermolecular CA-CA interactions.

The electrostatic behavior of titrating groups in alpha-sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the alpha-sarcin-2'-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, approximately 4.8, and 6.8 in the alpha-sarcin-2'-GMP complex, respectively. The pKa values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137). The absence of this interaction in alpha-sarcin would explain the lower pKa value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.

The solution structure of the antifungal protein (AFP, 51 residues, 4 disulfide bridges) from Aspergillus giganteus has been determined by using experimentally derived interproton distance constraints from nuclear magnetic resonance (NMR) spectroscopy. Complete sequence-specific proton assignments were obtained at pH 5.0 and 35 degrees C. A set of 834 upper limit distance constraints from nuclear Overhauser effect measurements was used as input for the calculation of structures with the program DIANA. An initial family of 40 structures calculated with no disulfide constraints was used to obtain information about the disulfide connectivities, which could not be determined by standard biochemical methods. Three possible disulfide patterns were selected and the corresponding disulfide constraints applied to generate a family of 20 DIANA conformers for each pattern. Following energy minimization, the average pairwise RMSD of the 20 conformers of each family is 1.01, 0.89, and 1.01 A for backbone atoms and 1.82, 1.74, and 1.81 A for all heavy atoms. One of these three families contains the disulfide bridge arrangement actually present in the solution structure of AFP. Although the three families fulfill the NMR constraints, one of the disulfide patterns considered (cysteine pairs 7-33, 14-40, 26-49, 28-51) is favored among the others on the basis of previous chemical studies. It thus probably corresponds to the actual pattern of disulfide bridges present in the protein, and the corresponding family represents the solution structure of AFP. The folding of AFP consists of five antiparallel beta strands connected in a -1, -1, +3, +1 topology and highly twisted, defining a small and compact beta barrel stabilized by four internal disulfide bridges. A cationic site formed by up to three lysine side chains adjacent to a hydrophobic stretch, both at the protein surface, may constitute a potential binding site for phospholipids which would be the basis of its biological function. On the other hand, a second, minor form of AFP has been detected. NMR data, together with results from mass spectrometry, chemical analysis, and sedimentation equilibrium, suggest that this species differs from the major form in the pairs of cysteines involved in the four disulfide bridges.