Calmodulin and troponin C structures studied by Fourier transform infrared spectroscopy: effects of Ca2+ and Mg2+ binding

Fourier transform infrared (FTIR) spectroscopy has been used to examine the conformationally sensitive amide I' bands of calmodulin and troponin C. These are observed to undergo a sequence of spectroscopic changes which reflect conformational rearrangements that take place when Ca2+ is bound. Calmodulin and troponin C show similar though not identical changes on Ca2+ binding, and the effect of Mg2+ on troponin C is quite different from that of Ca2+. Both proteins show absorption maxima in the amide I' region at 1644 cm-1 which is significantly lower in frequency than has been generally observed for proteins that contain a high percentage of alpha-helix. It is proposed that an unusually high proportion of the helices in the structures of these proteins are distorted from the normal alpha-helical configuration such that the carbonyl stretching frequencies are lowered. It is further proposed that the shift to lower frequency is due to backbone carbonyl groups in the distorted helices that form strong hydrogen bonds with solvent molecules. A decrease in intensity at 1654 cm-1, the normal frequency assignment for alpha-helical structure, is observed as Ca2+ binds to calmodulin and troponin C. This suggests that Ca2+ binding results in a net decrease in "normal" alpha-helix conformation. There is a corresponding increase in intensity of the band at 1644 cm-1, possibly due to an increase in distorted helix content, allowing for a net increase in helix consistent with circular dichroism estimates of the Ca2+-dependent changes in helix content in calmodulin.     

Comparative NMR studies on cardiac troponin C and a mutant incapable of binding calcium at site II

One- and two-dimensional NMR techniques were used to study both the influence of mutations on the structure of recombinant normal cardiac troponin C (cTnC3) and the conformational changes induced by Ca2+ binding to site II, the site responsible for triggering muscle contraction. Spin systems of the nine Phe and three Tyr residues were elucidated from DQF-COSY and NOESY spectra. Comparison of the pattern of NOE connectivities obtained from a NOESY spectrum of cTnC3 with a model of cTnC based on the crystal structure of skeletal TnC permitted sequence-specific assignment of all three Tyr residues, as well as Phe-101 and Phe-153. NOESY spectra and calcium titrations of cTnC3 monitoring the aromatic region of the 1H NMR spectrum permitted localization of six of the nine Phe residues to either the N- or C-terminal domain of cTnC3. Analysis of the downfield-shifted C alpha H resonances permitted sequence-specific assignment of those residues involved in the beta-strand structures which are part of the Ca(2+)-binding loops in both the N- and C-terminal domains of cTnC3. The short beta-strands in the N-terminal domain of cTnC3 were found to be present and in close proximity even in the absence of Ca2+ bound at site II. Using these assignments, we have examined the effects of mutating Asp-65 to Ala, CBM-IIA, a functionally inactive mutant which is incapable of binding Ca2+ at site II [Putkey, J.A., Sweeney, H. L., & Campbell, S. T. (1989) J. Biol. Chem. 264, 12370]. Comparison of the apo, Mg(2+)-, and Ca(2+)-bound forms of cTnC3 and CBM-IIA demonstrates that the inability of CBM-IIA to trigger muscle contraction is not due to global structural changes in the mutant protein but is a consequence of the inability of CBM-IIA to bind Ca2+ at site II. The pattern of NOEs between aromatic residues in the C-terminal domain is nearly identical in cTnC3 and CBM-IIA. Similar interresidue NOEs were also observed between Phe residues assigned to the N-terminal domain in the Ca(2+)-saturated forms of both cTnC3 and CBM-IIA. However, chemical shift changes were observed for the N-terminal Phe residues in CBM-IIA. This suggests that binding of Ca2+ to site II alters the chemical environment of the residues in the N-terminal hydrophobic cluster without disrupting the spatial relationship between the Phe residues located in helices A and D.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

A Refined Model of the Prototypical Salmonella SPI-1 T3SS Basal Body Reveals the Molecular Basis for Its Assembly

The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a “basal body”, a lock-nut structure spanning both bacterial membranes, and a “needle” that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.     

Insights into the molecular basis for the carbenicillinase activity of PSE-4 beta-lactamase from crystallographic and kinetic studies

PSE-4 is a class A beta-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild-type PSE-4 carbenicillinase has been determined to 1.95 A resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. A superposition of the PSE-4 structure with that of TEM-1 shows a rms deviation of 1.3 A for 263 Calpha atoms. Most carbenicillinases are unique among class A beta-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50-fold reduction in k(cat)/K(m) and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 A resolution with the wild-type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A beta-lactamase structures. Our molecular modeling studies suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin alpha-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild-type PSE-4 may be involved in accommodating this shift in the bound substrate position.     

Effect of divalent metal cations on the dimerization of OXA-10 and -14 class D beta-lactamases from Pseudomonas aeruginosa

The factors influencing the oligomerization state of OXA-10 and OXA-14 class D beta-lactamases in solution have been investigated. Both enzymes were found to exist as an equilibrium mixture of a monomer and dimer, with a K(d) close to 40 microM. The dimeric form was stabilized by divalent metal cations. The ability of different metal ions to stabilize the dimer was in the following order: Cd(2+) > Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) > Mn(2+) > Ca(2+) > Mg(2+). The apparent K(d)s describing the binding of Zn(2+) and Cd(2+) cations to the OXA-10 dimer were 7.8 and 5.7 microM, respectively. The metal ions had a profound effect on the thermal stability of the protein complex observed by differential scanning calorimetry. The enzyme showed a sharp transition with a T(m) of 58.7 degrees C in the absence of divalent cations, and an equally sharp transition with a T(m) of 78.4 degrees C in the presence of a saturating concentration of the divalent cation. The thermal transition observed at intermediate concentrations of divalent metal ions was rather broad and lies between these two extremes of temperature. The equilibrium between the monomer and dimer is dependent on pH, and the optimum for the formation of the dimer shifted from pH 6.0 in the absence of divalent cations to pH 7.5 at saturating concentrations. The beta-lactamase activity increased approximately 2-fold in the presence of saturating concentrations of zinc and cadmium ions. Reaction with beta-lactams caused a shift in the equilibrium toward monomer formation, and thus an apparent inactivation, but the divalent cations protected against this effect.     

Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog

Sialic acid terminates oligosaccharide chains on mammalian and microbial cell surfaces, playing critical roles in recognition and adherence. The enzymes that transfer the sialic acid moiety from cytidine-5'-monophospho-N-acetyl-neuraminic acid (CMP-NeuAc) to the terminal positions of these key glycoconjugates are known as sialyltransferases. Despite their important biological roles, little is understood about the mechanism or molecular structure of these membrane-associated enzymes. We report the first structure of a sialyltransferase, that of CstII from Campylobacter jejuni, a highly prevalent foodborne pathogen. Our structural, mutagenesis and kinetic data provide support for a novel mode of substrate binding and glycosyl transfer mechanism, including essential roles of a histidine (general base) and two tyrosine residues (coordination of the phosphate leaving group). This work provides a framework for understanding the activity of several sialyltransferases, from bacterial to human, and for the structure-based design of specific inhibitors.     

Crystal structure of LexA: a conformational switch for regulation of self-cleavage

LexA repressor undergoes a self-cleavage reaction. In vivo, this reaction requires an activated form of RecA, but it occurs spontaneously in vitro at high pH. Accordingly, LexA must both allow self-cleavage and yet prevent this reaction in the absence of a stimulus. We have solved the crystal structures of several mutant forms of LexA. Strikingly, two distinct conformations are observed, one compatible with cleavage, and the other in which the cleavage site is approximately 20 A from the catalytic center. Our analysis provides insight into the structural and energetic features that modulate the interconversion between these two forms and hence the rate of the self-cleavage reaction. We suggest RecA activates the self-cleavage of LexA and related proteins through selective stabilization of the cleavable conformation.     

Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus

The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad-spectrum beta-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Because of its low affinity for beta-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at beta-lactam concentrations that inhibit the beta-lactam-sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 A resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefin, penicillin G and methicillin allow, for the first time, a comparison of an apo and acylated resistant PBP. An analysis of the PBP2a active site in these forms reveals the structural basis of its resistance and identifies features in newly developed beta-lactams that are likely important for high affinity binding.     

Crystal structure of the class D beta-lactamase OXA-10

We report the crystal structure of a class D beta-lactamase, the broad spectrum enzyme OXA-10 from Pseudomonas aeruginosa at 2.0 A resolution. There are significant differences between the overall fold observed in this structure and those of the evolutionarily related class A and class C beta-lactamases. Furthermore, the structure suggests the unique, cation mediated formation of a homodimer. Kinetic and hydrodynamic data shows that the dimer is a relevant species in solution and is the more active form of the enzyme. Comparison of the molecular details of the active sites of the class A and class C enzymes with the OXA-10 structure reveals that there is no counterpart in OXA-10 to the residues proposed to act as general bases in either of these enzymes (Glu 166 and Tyr 150, respectively). Our structures of the native and chloride inhibited forms of OXA-10 suggest that the class D enzymes have evolved a distinct catalytic mechanism for beta-lactam hydrolysis. Clinical variants of OXA-10 are also discussed in light of the structure.