Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release

Abstract

Calmodulin (CaM) expression is upregulated upon HIV-1 infection and interacts with proteins involved in viral processing, including the multifunctional HIV-1 MA protein. We present here the results of studies utilizing small-angle neutron scattering with contrast variation that, when considered in the light of earlier fluorescence and NMR data, show CaM binds MA in an extended open-clamp conformation via interactions with two tryptophans that are widely spaced in sequence and space. The interaction requires a disruption of the MA tertiary fold such that MA becomes highly extended in a long snakelike conformation. The CaM-MA interface is extensive, covering ~70% of the length of the MA such that regions known to be important in MA interactions with critical binding partners would be impacted. The CaM conformation is semiextended and as such is distinct from the classical CaM-collapse about short α-helical targets. NMR data show that upon dissociation of the CaM-MA complex, either by the removal of Ca²⁺ or increasing ionic strength, MA reforms its native tertiary contacts. Thus, we observe a high level of structural plasticity in MA that may facilitate regulation of its activities via intracellular Ca²⁺-signaling during viral processing.

Figure 1

Scattering data for CaM-MA. (a) I(q) versus q and (b) P(r) versus r. Symbol key: SAXS data (□), and SANS data for CaM-MA in 19% D₂O (○), 44% D₂O (▵), 88% (▿), and 100% D₂O (⋄). The solid lines in a are the fits of the MONSA-derived model in Fig. 3. The I(q) profiles have been offset on the vertical scale for clarity. The P(r) profiles are on a relative scale that accounts for the contrast of each scattering profile.

Figure 2

Comparisons of CaM in CaM-MA with CaM-peptide structures. (a) 44% D₂O I(q) versus q for CaM-MA (▵) with CRYSON fits of the crystal structures of CaM (PDB:1CLL) (dotted line), and CaM taken from its structures in its complexes with Munc13 (PDB:2KDU) (solid line) and skMLCK (PDB:2BBM) (dashed line). (b) P(r) versus r for the experimental and modeled profiles shown in panel a using the same key.

Figure 3

MONSA-derived shape model for CaM-MA.

Figure 4

Two-dimensional ¹⁵N-¹H-HSQC spectra from CaM-MA. (a) ¹⁵N-MA only; (b) ¹⁵N-MA-CaM complex in 10 mM NaPO₄ (pH 5.5), 5 mM CaCl₂, 1 mM TCEP; (c and d) the ¹⁵N-MA-CaM complex after titration with either KCl (to 500 mM) or EGTA (10 mM), respectively, showing many peaks corresponding to folded MA have been restored; (e) ¹⁵N-MA in 6 M GuHCl, and (f) ¹⁵N-MA after removal of 6 M GuHCl.

Figure 5

Schematic of CaM-MA interaction. In the presence of Ca²⁺ and at low ionic strength, CaM binds MA in an open-clamp, semiextended bidentate conformation resulting in a dramatic extension of MA. Release of MA, which can be induced either by increasing ionic strength or loss of Ca²⁺, results in reformation of the native, free MA structure.