Energetics and mechanisms of folding and flipping the myristoyl switch in the β-trefoil protein, hisactophilin

Abstract

Myristoylation, the covalent linkage of a saturated, C 14 fatty acyl chain to the N-terminal glycine in a protein, plays a vital role in reversible membrane binding and signaling by the modified proteins. Currently, little is known about the effects of myristoylation on protein folding and stability, or about the energetics and molecular mechanisms of switching involving states with sequestered versus accessible myristoyl group. Our analysis of these effects in hisactophilin, a histidine-rich protein that binds cell membranes and actin in a pH-dependent manner, shows that myristoylation significantly increases hisactophilin stability, while also markedly increasing global protein folding and unfolding rates. The switching between sequestered and accessible states is pH dependent, with an apparent pK switch of 6.95, and an apparent free energy change of 2.0 kcal·mol −1 . The myristoyl switch is linked to the reversible uptake of ∼1.5 protons, likely by histidine residues. This pH dependence of switching appears to be the physical basis of the sensitive, pH-dependent regulation of membrane binding observed in vivo. We conclude that an increase in protein stability upon modification and burial of the attached group is likely to occur in numerous proteins modified with fatty acyl or other hydrophobic groups, and that the biophysical effects of such modification are likely to play an important role in their functional switches. In addition, the increased global dynamics caused by myristoylation of hisactophilin reveals a general mechanism whereby hydrophobic moieties can make nonnative interactions or relieve strain in transition states, thereby increasing the rates of interconversion between different states. thermodynamic cycle | switch dynamics | switch energetics M yristoylation is a common cotranslational modification found in ∼0.5-0.8% of eukaryotic proteins (1). This modification involves the covalent linkage of a saturated C 14 fatty acyl chain to the N-terminal glycine residue in a protein (1). Myristoylated proteins play vital roles in many biological processes and commonly undergo reversible switches. The "flipping" of myristoyl switches typically involves interconversion between a myristoyl-sequestered state, myr seq , where the myristoyl group is located in a hydrophobic binding pocket within the protein, and a myristoyl-accessible state, myr acc , where the myristoyl group is available for binding to membranes or other proteins. Switching may be associated with relatively large or subtle structural and/or dynamic changes in the myristoylated protein (2, 3). It can also be regulated by binding of various ligands (e.g., H þ , Ca 2þ , GTP, or regulatory protein) (3-5). Some examples of proteins that undergo myristoyl switching include: Ca 2þ -dependent recoverin, which mediates photoresponses in the retina (3); Ca 2þ -dependent guanylate cyclase activating protein (GCAP), which regulates the function of guanylate cyclase (2, 6); oligomerization-dependent HIV-1 Gag, which orchestrates HIV-1 viral proliferat...

β-Bulge triggers route-switching on the functional landscape of interleukin-1β

“…IL-1β contains three trefoil subunits (βββ-loop-β), in which six β-strands (strands 1,4,5,8,9,12) form the barrel and six β-strands (strands 2, 3, 6, 7, 10, 11) form a hairpin cap (12)(13)(14). The protein populates a well-defined structured intermediate on the folding pathway in which the central strands (β-strands 6-10) form early followed by packing of the N-and C-terminal strands to close the β-barrel (15,16).…”

“…The question remains, what factors control the route selection for folding? There is speculation that functional residues are a hindrance to folding and may add heterogeneity and roughness to the landscape (2)(3)(4)(5)(6)(7)(8). These residues need to be conserved for the protein to function correctly and therefore cannot be optimized for folding.…”

Nonnative interactions regulate folding and switching of myristoylated protein

“…The dominant role of native interactions is manifested by the funnel-shaped energy landscape for folding that suggests folding is robust and an efficient process. The information stored in the native topology may, however, be tuned by various factors such as confining the protein in a small space, crowding agents, or conjugating the protein to other biomolecules [e.g., oligosaccharides (5) or fatty acyl chains such as myristoyl (6)]. In addition to manipulating folding characteristics by modifications or environmental conditions, nonnative interactions, which are by definition in conflict with the native state, may decorate the folding funnel (7,8) by increasing energetic frustration (9) between interactions and therefore landscape roughness.…”

“…Investigations of several proteins have reported evidence for nonnative interactions that assist, rather than hinder, folding (10)(11)(12)(13)(14), and more importantly they may also support function (6,15,16) by assisting conformational changes. Residues in functional sites in proteins have been implicated in causing geometric frustration (17) or increasing localized energetic frustration (16).…”

“…The myristoyl increases hisactophilin stability with an apparent pK a of 6.95 as it switches between a "sequestered" state at high pH, where the myristoyl is buried in the protein core (Fig. 1A), and an "accessible" state at low pH for membrane binding (6). Concomitantly, the myristoyl markedly accelerates both folding and unfolding kinetics, and undergoes rapid native-state switching, implicating the long and flexible hydrophobic myristoyl chain in creating strain in the native state and forming nonnative interactions during folding and switching.…”