Molecular recognition of an ADP-ribosylating Clostridium botulinum C3 exoenzyme by RalA GTPase.

C3 exoenzymes (members of the ADP-ribosyltranferase family) are produced by Clostridium botulinum (C3bot1 and -2), Clostridium limosum (C3lim), Bacillus cereus (C3cer), and Staphylococcus aureus (C3stau1-3). These exoenzymes lack a translocation domain but are known to specifically inactivate Rho GTPases in host target cells. Here, we report the crystal structure of C3bot1 in complex with RalA (a GTPase of the Ras subfamily) and GDP at a resolution of 2.66 A. RalA is not ADP-ribosylated by C3 exoenzymes but inhibits ADP-ribosylation of RhoA by C3bot1, C3lim, and C3cer to different extents. The structure provides an insight into the molecular interactions between C3bot1 and RalA involving the catalytic ADP-ribosylating turn-turn (ARTT) loop from C3bot1 and helix alpha4 and strand beta6 (which are not part of the GDP-binding pocket) from RalA. The structure also suggests a molecular explanation for the different levels of C3-exoenzyme inhibition by RalA and why RhoA does not bind C3bot1 in this manner.

C3 exoenzyme from Clostridium botulinum: structure of a tetragonal crystal form and a reassessment of NAD-induced flexure.

C3 exoenzyme from Clostridium botulinum (C3bot1) ADP-ribosylates and thereby inactivates Rho A, B and C GTPases in mammalian cells. The structure of a tetragonal crystal form has been determined by molecular replacement and refined to 1.89 A resolution. It is very similar to the apo structures determined previously from two different monoclinic crystal forms. An objective reassessment of available apo and nucleotide-bound C3bot1 structures indicates that, contrary to a previous report, the protein possesses a rigid core formed largely of beta-strands and that the general flexure that accompanies NAD binding is concentrated in two peripheral lobes. Tetragonal crystals disintegrate in the presence of NAD, most likely because of disruption of essential crystal contacts.

Structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin I-converting enzyme.

Angiotensin converting enzyme (ACE) plays a critical role in the circulating or endocrine renin-angiotensin system (RAS) as well as the local regulation that exists in tissues such as the myocardium and skeletal muscle. Here we report the high-resolution crystal structures of testis ACE (tACE) in complex with the first successfully designed ACE inhibitor captopril and enalaprilat, the Phe-Ala-Pro analogue. We have compared these structures with the recently reported structure of a tACE-lisinopril complex [Natesh et al. (2003) Nature 421, 551-554]. The analyses reveal that all three inhibitors make direct interactions with the catalytic Zn(2+) ion at the active site of the enzyme: the thiol group of captopril and the carboxylate group of enalaprilat and lisinopril. Subtle differences are also observed at other regions of the binding pocket. These are compared with N-domain models and discussed with reference to published biochemical data. The chloride coordination geometries of the three structures are discussed and compared with other ACE analogues. It is anticipated that the molecular details provided by these structures will be used to improve the binding and/or the design of new, more potent domain-specific inhibitors of ACE that could serve as new generation antihypertensive drugs.

The crystal structure of C3stau2 from Staphylococcus aureus and its complex with NAD.

The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase that ADP-ribosylates not only RhoA-C but also RhoE/Rnd3. In this study we have crystallized and determined the structure of C3stau2 in both its native form and in complex with NAD at 1.68- and 2.02-A resolutions, respectively. The topology of C3stau2 is similar to that of C3bot1 from Clostridium botulinum (with which it shares 35% amino acid sequence identity) with the addition of two extra helices after strand beta1. The native structure also features a novel orientation of the catalytic ARTT loop, which approximates the conformation seen for the "NAD bound" form of C3bot1. C3stau2 orients NAD similarly to C3bot1, and on binding NAD, C3stau2 undergoes a clasping motion and a rearrangement of the phosphate-nicotinamide binding loop, enclosing the NAD in the binding site. Comparison of these structures with those of C3bot1 and related toxins reveals a degree of divergence in the interactions with the adenine moiety among the ADP-ribosylating toxins that contrasts with the more conserved interactions with the nicotinamide. Comparison with C3bot1 gives some insight into the different protein substrate specificities of these enzymes.

The crystal structure of eosinophil cationic protein in complex with 2',5'-ADP at 2.0 A resolution reveals the details of the ribonucleolytic active site.

Eosinophil cationic protein (ECP) is a component of the eosinophil granule matrix. It shows marked toxicity against helminth parasites, bacteria single-stranded RNA viruses, and host epithelial cells. Secretion of human ECP is related to eosinophil-associated allergic, asthmatic, and inflammatory diseases. ECP belongs to the pancreatic ribonuclease superfamily of proteins, and the crystal structure of ECP in the unliganded form (determined previously) exhibited a conserved RNase A fold [Boix, E., et al. (1999) Biochemistry 38, 16794-16801]. We have now determined a high-resolution (2.0 A) crystal structure of ECP in complex with adenosine 2',5'-diphosphate (2',5'-ADP) which has revealed the details of the ribonucleolytic active site. Residues Gln-14, His-15, and Lys-38 make hydrogen bond interactions with the phosphate at the P(1) site, while His-128 interacts with the purine ring at the B(2) site. A new phosphate binding site, P(-)(1), has been identified which involves Arg-34. This study is the first detailed structural analysis of the nucleotide recognition site in ECP and provides a starting point for the understanding of its substrate specificity and low catalytic efficiency compared with that of the eosinophil-derived neurotoxin (EDN), a close homologue.