High resolution x-ray analysis of two mutants of a curaremimetic snake toxin.

A previous mutational analysis of erabutoxin a (Ea), a curaremimetic toxin from sea snake venom, showed that the substitutions S8G and S8T caused, respectively, 176-fold and 780-fold affinity decreases for the nicotinic acetylcholine receptor (AchR). In view of the fact that the side-chain of Ser8 is buried in the wild-type toxin, we wondered whether these affinity changes reflect a direct binding contribution of S8 to the receptor and/or conformational changes that could have occurred in Ea as a result of the introduced mutations. To approach this question, we solved X-ray structures of the two mutants S8G and S8T at high resolution (0.18 nm and 0.17 nm, with R factors of 18.0% and 17.9%, respectively). The data show that none of the mutations significantly modified the toxin structure. Even within the site where the toxin binds to the receptor the backbone conformation remained unchanged. Therefore, the low affinities of the mutants S8T and S8G cannot be explained by a large conformational change of the toxin structure. Although we cannot exclude the possibility that undetectable structural changes have occurred in the toxin mutants, our data support the view that, although buried between loop I and II, S8 is part of the functional epitope of the toxin.

Crystal structures of alpha-mercaptoacyldipeptides in the thermolysin active site: structural parameters for a Zn monodentation or bidentation in metalloendopeptidases.

Three alpha-mercaptoacyldipeptides differing essentially in the size of their C-terminal residues have been crystallized in the thermolysin active site. A new mode of binding was observed for 3 [HS-CH(CH(2)Ph)CO-Phe-Tyr] and 4 [HS-CH((CH(2))(4)CH(3))CO-Phe-Ala], in which the mercaptoacyl moieties act as bidentates with Zn-S and Zn-O distances of 2.3 and 2.4 A, respectively, the side chains fitting the S(1), S(1)', and S(2)' pockets. Moreover, a distance of 3.1 A between the sulfur atom and the OE1 of Glu(143) suggests that they are H-bonded and that one of these atoms is protonated. This H-bond network involving Glu(143), the mercaptoacyl group of the inhibitor, and the Zn ion could be considered a "modified" transition state mimic of the peptide bond hydrolysis. Due to the presence of the hindering (5-phenyl)proline, the inhibitor HS-CH(CH(2)Ph)CO-Gly-(5-Ph)Pro (2) interacts through the usual Zn monodentation via the thiol group and occupancy of S(1)' and S(2)' subsites by the aromatic moieties, the proline ring being outside the active site. The inhibitory potencies are consistent with these structural data, with higher affinities for 3 (4.2 x 10(-)(8) M) and 4 (4.8 x 10(-)(8) M) than for 2 (1.2 x 10(-)(6) M). The extension of the results, obtained with thermolysin being considered as the model of physiological zinc metallopeptidases, allows inhibitor-recognition modes for other peptidases, such as angiotensin converting enzyme and neutral endopeptidase, to be proposed and opens interesting possibilities for the design of new classes of inhibitors.

Structures of escherichia coli CMP kinase alone and in complex with CDP: a new fold of the nucleoside monophosphate binding domain and insights into cytosine nucleotide specificity.

BACKGROUND: . Nucleoside monophosphate kinases (NMP kinases) catalyze the reversible transfer of a phosphoryl group from a nucleoside triphosphate to a nucleoside monophosphate. Among them, cytidine monophosphate kinase from Escherichia coli has a striking particularity: it is specific for CMP, whereas in eukaryotes a unique UMP/CMP kinase phosphorylates both CMP and UMP with similar efficiency. RESULTS: . The crystal structure of the CMP kinase apoenzyme from E. coli was solved by single isomorphous replacement and refined at 1.75 A resolution. The structure of the enzyme in complex with CDP was determined at 2.0 A resolution. Like other NMP kinases, the protein contains a central parallel beta sheet, the strands of which are connected by alpha helices. The enzyme differs from other NMP kinases in the presence of a 40-residue insert situated in the NMP-binding (NMPbind) domain. This insert contains two domains: one comprising a three-stranded antiparallel beta sheet, the other comprising two alpha helices. CONCLUSIONS: . Two features of the CMP kinase from E. coli have no equivalent in other NMP kinases of known structure. Firstly, the large NMPbind insert undergoes a CDP-induced rearrangement: its beta-sheet domain moves away from the substrate, whereas its helical domain comes closer to it in a motion likely to improve the protection of the active site. Secondly, residues involved in CDP recognition are conserved in CMP kinases and have no counterpart in other NMP kinases. The structures presented here are the first of a new family of NMP kinases specific for CMP.

Solubility diagram of the Rhodobacter sphaeroides reaction center as a function of PEG concentration.

In order to quantify the effect of polyethylene glycol 4000 (PEG) on the solubility of an integral membrane protein, we have crystallized the photochemical reaction center from Rhodobacter sphaeroides Y by batch method on a large range of PEG. The measurement of the solubility diagram display a semi-logarithmic dependence of solubility versus PEG concentration. Comparison of our results with previously published ones [Odahara, T., Ataka, M. and Katsura, M. (1994) Acta Cryst. D50, 639-642] suggests a notable effect of additional 1,2,3-heptane-triol and/or temperature on photochemical reaction center solubility.

Structure of the photochemical reaction centre of a spheroidene-containing purple-bacterium, Rhodobacter sphaeroides Y, at 3 A resolution.

The crystal structure of the photochemical reaction centre from Rhodobacter sphaeroides Y, a carotenoid-containing wild-type purple bacterium, has been determined at 3 A resolution. This membrane complex consists of three subunits (281, 307 and 260 residues, respectively) and ten cofactors. It was crystallized in presence of beta-D-octylglucoside. The crystals are orthorhombic with unit-cell dimensions, a = 143.7, b = 139.8, c = 78.7 A, space group P2(1)2(1)2(1) with four molecules in the unit cell. Refinement of the structure by X-PLOR and manual reconstructions yielded an R value of 22.1% for 19630 reflections between 7 and 3 A. The secondary structure is highly homologous to those determined for Rhodopseudomonas viridis (Protein Data Bank entry 1PRC) and Rhodobacter sphaeroides R26 (Protein Data Bank entry 4RCR) reaction centres. In the latter two structures one Fe(2+) ion located between the two quinones is coordinated by four histidines and one glutamic acid. In the Rhodobacter sphaeroides Y structure, Mn(2+) occupies the same position with identical ligands and geometry. The carotenoid conformation which is a non-planar 15-15'-cis spheroidene molecule in our structure differs from the 13-14-cis 2,4-dihydroneusporene in the Rhodopseudomonas viridis structure.