Nonnative interactions regulate folding and switching of myristoylated protein

Abstract

We present an integrated experimental and computational study of the molecular mechanisms by which myristoylation affects protein folding and function, which has been little characterized to date. Myristoylation, the covalent linkage of a hydrophobic C14 fatty acyl chain to the N-terminal glycine in a protein, is a common modification that plays a critical role in vital regulated cellular processes by undergoing reversible energetic and conformational switching. Coarse-grained folding simulations for the model pH-dependent actin-and membrane-binding protein hisactophilin reveal that nonnative hydrophobic interactions of the myristoyl with the protein as well as nonnative electrostatic interactions have a pronounced effect on folding rates and thermodynamic stability. Folding measurements for hydrophobic residue mutations of hisactophilin and atomistic simulations indicate that the nonnative interactions of the myristoyl group in the folding transition state are nonspecific and robust, and so smooth the energy landscape for folding. In contrast, myristoyl interactions in the native state are highly specific and tuned for sensitive control of switching functionality. Simulations and amide hydrogen exchange measurements provide evidence for increases as well as decreases in stability localized on one side of the myristoyl binding pocket in the protein, implicating strain and altered dynamics in switching. The effects of folding and function arising from myristoylation are profoundly different from the effects of other post-translational modifications.coarse-grained simulation | funnel landscape | β-trefoil P rotein folding is governed by various physicochemical forces that bias the native state, which for many proteins is also the functional state, over the many alternative nonnative states. The network of native interactions has been found in many cases to be sufficient to capture the folding mechanism and kinetics of proteins (1, 2). The discrimination between native and nonnative interactions is the foundation of the principle of minimal frustration (3) and explains the power of native topology-based models in studying folding biophysics (4). 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. The degree of roughness, which affects the trapping of the protein in nonnative states, depends on the particular sequence of the pr...

Understanding the Folding-Function Tradeoff in Proteins

“…The myristoyl chain switches between an exposed state which enables membrane binding and a state where it is buried in the β-barrel of the fold (cartoon shown in Fig. 8) [43], [45]. HIS has a lower folding barrier than the FM (Fig.…”

“… (a) Structure of HIS (orange) with key myristoyl binding residues [43], [45] marked in grey. In order to accommodate and bind the myristoyl chain the grey residues do not have the contacts marked in green (M) with the green residues.…”

“…Specifically, we compare the folding of the FM to that of two WT proteins IL-1β [30] and hisactophilin (HIS) [38]. We choose these proteins for two reasons: (a) Folding experiments [19]–[20], [39]–[43] corroborate the folding routes and rates found in MD simulations [18], [31], [44], [45] of these proteins. These simulations were performed using SBMs similar to those used in this article.…”

How Well Does a Funneled Energy Landscape Capture the Folding Mechanism of Spectrin Domains?

“…The idea of combining a non-native potential with a Gō model is not novel and has been pioneered by others. 8,46–50 It is important to note that residue pairs may be either mutually attractive or repulsive in this potential, depending on their types. The free energy surfaces for this modified model are shown in 2B,D,F.…”

Chemical physics of protein folding

“…On the computational side, although effective theories of folding can often use simplified models that make interesting and surprising predictions (20,21), many questions of detail can now be answered satisfactorily with fully atomistic simulations that challenge computational power to the limit (22)(23)(24).…”