Solution Structures of the C-Terminal Domain of Cardiac Troponin C Free and Bound to the N-Terminal Domain of Cardiac Troponin I

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33−80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81−161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81−161) both free and bound to cTnI(33−80). No significant differences were observed between secondary structural elements determined for free and cTnI(33−80)-bound cTnC(81−161). We have determined solution structures of Ca2+-saturated cTnC(81−161) free and bound to cTnI(33−80). While the tertiary structure of cTnC(81−161) is qualitatively similar to that observed free in solution, the binding of cTnI(33−80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33−80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33−80), onto the C-terminal cTnC structure. The binding interface for cTnI(33−80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex

Revealing how an adenylate cyclase toxin uses bait and switch tactics in its activation.

Dissecting how bacterial pathogens escape immune destruction and cause respiratory infections in humans is a work in progress. One tactic employed by microbes is to use bacterial adenylate cyclase toxins (ACTs) to disarm immune cells and disrupt cellular signaling in host cells, which facilitates the infection process. Several clinically significant pathogens, such as Bacillus anthracis and Bordetella pertussis, have ACTs that are stimulated by an activator protein in human cells. Research has shown that these bacterial ACTs have evolved distinct ways of controlling their activities, but our understanding of how the B. pertussis ACT does this is limited. In a recent study, O'Brien and colleagues provide new and exciting evidence demonstrating that the regulation of B. pertussis ACT involves conformational switching between flexible and rigid states, which is triggered upon binding the host activator protein. This study increases our knowledge of how bacterial ACTs are unique enzymes, representing a potentially novel class of drug targets that may open new pathways to combat reemerging infectious diseases

Model depicting the dispositions of structural elements important in the CaM-dependent activation of two bacterial ACTs.

<p>(A) In the absence of CaM, the helical domain of EF (green rectangle) limits the accessibility of the catalytic core (orange rectangle), which contains the CaM-binding determinant (cyan ellipse). (B) When CaM (depicted by a pink sphere representing the N-terminal domain and a red sphere denoting the C-terminal domain) binds in the presence of 2–4 Ca²⁺ ions (yellow spheres), the helical domain reorients, exposing the catalytic site (yellow star) of EF. (C) Surprisingly, two key structural elements of CyaA-ACD are disordered in the absence of CaM (gray and cyan traces), while the catalytic core (orange) is unstably folded (flexibility is depicted by wavy black outline). (D) CyaA-ACD, which lacks a helical domain, is inactive in the absence of CaM, presumably due to the lack of a stable catalytic site. In the presence of 4Ca²⁺-CaM, the domain involved in C-terminal CaM-binding (cyan ellipse) and another key region (gray ellipse with black arrow) become stably folded with increased rigidity. Furthermore, the catalytic core (orange rectangle with solid black line) is stabilized by CaM-binding and readily converts ATP to cAMP. ACT, adenylate cyclase toxin; ATP, adenosine triphosphate; CaM, calmodulin; cAMP, cyclic adenosine monophosphate; CyaA-ACD, adenylate cyclase domain of CyaA; EF, edema factor.</p

Site I Inactivation Impacts Calmodulin Calcium Binding and Activation of Bordetella pertussis Adenylate Cyclase Toxin

Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable α-helical content to CaMWt, and only modest differences in α-helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C-terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N-terminal CaM and CyaA-ACD

Structural and functional analysis of the Acinetobacter baumannii BlsA photoreceptor and regulatory protein.

The Acinetobacter baumannii BlsA photoreceptor has an N-terminal (NT) BLUF domain and a C-terminal (CT) amino acid sequence with no significant homology to characterized bacterial proteins. In this study, we tested the biological role of specific residues located in these BlsA regions. Site-directed mutagenesis, surface motility assays at 24°C and protein overexpression showed that residues Y7, Q51 and W92 are essential for not only light-regulated motility, but also BlsA's solubility when overexpressed in a heterologous host. In contrast, residues A29 and F32, the latter representing a difference when compared with other BLUF-containing photoreceptors, do not play a major role in BlsA's biological functions. Analysis of the CT region showed that the deletion of the last five BlsA residues has no significant effect on the protein's light-sensing and motility regulatory functions, but the deletion of the last 14 residues as well as K144E and K145E substitutions significantly alter light-regulated motility responses. In contrast to the NT mutants, these CT derivatives were overexpressed and purified to homogeneity to demonstrate that although these mutations do not significantly affect flavin binding and photocycling, they do affect BlsA's photodynamic properties. Notably, these mutations map within a potential fifth α-helical component that could play a role in predicted interactions between regulatory partners and BlsA, which could function as a monomer according to gel filtration data. All these observations indicate that although BlsA shares common structural and functional properties with unrelated photoreceptors, it also exhibits unique features that make it a distinct BLUF photoreceptor

Calcium-Dependent Interaction Occurs between Slow Skeletal Myosin Binding Protein C and Calmodulin

Myosin binding protein C (MyBP-C) is a multi-domain protein that participates in the regulation of muscle contraction through dynamic interactions with actin and myosin. Three primary isoforms of MyBP-C exist: cardiac (cMyBP-C), fast skeletal (fsMyBP-C), and slow skeletal (ssMyBP-C). The N-terminal region of cMyBP-C contains the M-motif, a three-helix bundle that binds Ca2+-loaded calmodulin (CaM), but less is known about N-terminal ssMyBP-C and fsMyBP-C. Here, we characterized the conformation of a recombinant N-terminal fragment of ssMyBP-C (ssC1C2) using differential scanning fluorimetry, nuclear magnetic resonance, and molecular modeling. Our studies revealed that ssC1C2 has altered thermal stability in the presence and absence of CaM. We observed that site-specific interaction between CaM and the M-motif of ssC1C2 occurs in a Ca2+-dependent manner. Molecular modeling supported that the M-motif of ssC1C2 likely adopts a three-helix bundle fold comparable to cMyBP-C. Our study provides evidence that ssMyBP-C has overlapping structural determinants, in common with the cardiac isoform, which are important in controlling protein–protein interactions. We shed light on the differential molecular regulation of contractility that exists between skeletal and cardiac muscle