Solution X-ray Scattering Reveals a Novel Structure of Calmodulin Complexed with a Binding Domain Peptide from the HIV-1 Matrix Protein p17

Abstract

The solution structures of complexes between calcium-saturated calmodulin (Ca (2+)/CaM) and a CaM-binding domain of the HIV-1 matrix protein p17 have been determined by small-angle X-ray scattering with use of synchrotron radiation as an intense and stable X-ray source. We used three synthetic peptides of residues 11-28, 26-47, and 11-47 of p17 to demonstrate the diversity of CaM-binding conformation. Ca (2+)/CaM complexed with residues 11-28 of p17 adopts a dumbbell-like structure at a molar ratio of 1:2, suggesting that the two peptides bind each lobe of CaM, respectively. Ca (2+)/CaM complexed with residues 26-47 of p17 at a molar ratio of 1:1 adopts a globular structure similar to the NMR structure of Ca (2+)/CaM bound to M13, which adopted a compact globular structure. In contrast to these complexes, Ca (2+)/CaM binds directly with both CaM-binding sites of residues 11-47 of p17 at a molar ratio of 1:1, which induces a novel structure different from known structures previously reported between Ca (2+)/CaM and peptide. A tertiary structural model of the novel structure was constructed using the biopolymer module of Insight II 2000 on the basis of the scattering data. The two domains of CaM remain essentially unchanged upon complexation. The hinge motions, however, occur in a highly flexible linker of CaM, in which the electrostatic residues 74Arg, 78Asp, and 82Glu interact with N-terminal electrostatic residues of the peptide (residues 12Glu, 15Arg, and 18Lys). The acidic residues in the N-terminal domain of CaM interact with basic residues in a central part of the peptide, thereby enabling the central part to change the conformations, while an acidic residue in the C-terminal domain interacts with two basic residues in the two helical sites of the peptide. The overall structure of the complex adopts an extended structure with the radius of gyration of 20.5 A and the interdomain distance of 34.2 A. Thus, the complex is principally stabilized by electrostatic interactions. The hydrophobic patches of Ca (2+)/CaM are not responsible for the binding with the hydrophobic residues in the peptide, suggesting that CaM plays a role to sequester the myristic acid moiety of p17.

Calmodulin Disrupts the Structure of the HIV-1 MA Protein

“…Although a similar number of chemical shift perturbations were expected from residues in the C-terminal lobe, relatively few were actually observed to change upon MA binding, comprising only two (L105 and T110) out of thirteen chemical shifts unambiguously assigned to residues in that lobe. The lack of perturbed chemical shifts in the C-terminal lobe may be due to the relative scarcity of assignments on our NMR experiment for residues in this region that have previously been identified to form contacts with target molecules (F92, V108, M109, F141, M144, and M14524; 41). It is also possible that the CaM-MA interaction is principally mediated by the N-terminal lobe, and we note that the proposed model by Izumi and colleagues also shows a disproportionate number of MA contacts from that lobe.…”

“…Izumi and colleagues41 have previously generated a model for the Ca 2+ -CaM-MA interaction using MA peptides corresponding to residues 11–47 and proposed that a group of charged residues in CaM on the N-terminal lobe and within the central helix form direct electrostatic contacts with MA. They proposed a novel interaction that excluded the involvement of CaM’s hydrophobic clefts that are typically responsible for CaM-target binding, and rather suggested that CaM could interact with MA without the involvement of the large hydrophobic residues internal to the MA core that would require a conformational change in the MA structure.…”

Solution Structure of Calmodulin Bound to the Binding Domain of the HIV-1 Matrix Protein

“…In vivo and in vitro studies revealed that CaM interacts with additional HIV-1 proteins like Nef, Tat, and gp160 (27,34,37,52,53,56). Small-angle x-ray scattering studies have provided a global picture of CaM bound to full-length MA (57) or to short peptides derived from the MA protein (58). Our laboratory (59) and others (57,60) have shown that CaM binds directly to the MA protein in a calcium-dependent manner.…”

Role of the HIV-1 Matrix Protein in Gag Intracellular Trafficking and Targeting to the Plasma Membrane for Virus Assembly

“…These studies have prompted others to conduct SAXS experiments to elucidate the global features of CaM bound to short peptides derived from the MA protein (Izumi et al, 2008). SAXS studies conducted on CaM complexed with full-length MA shed light on the global features of the complex (Chow et al, 2010).…”