Primary Contact Sites in Intrinsically Unstructured Proteins:  The Case of Calpastatin and Microtubule-Associated Protein 2

Abstract

Intrinsically unstructured proteins (IUPs) exist in a disordered conformational state, often considered to be equivalent with the random-coil structure. We challenge this simplifying view by limited proteolysis, circular dichroism (CD) spectroscopy, and solid-state (1)H NMR, to show short- and long-range structural organization in two IUPs, the first inhibitory domain of calpastatin (CSD1) and microtubule-associated protein 2c (MAP2c). Proteases of either narrow (trypsin, chymotrypsin, and plasmin) or broad (subtilisin and proteinase K) substrate specificity, applied at very low concentrations, preferentially cleaved both proteins in regions, i.e., subdomains A, B, and C in CSD1 and the proline-rich region (PRR) in MAP2c, that are destined to form contacts with their targets. For CSD1, nonadditivity of the CD spectra of its two halves and suboptimal hydration of the full-length protein measured by solid-state NMR demonstrate that long-range tertiary interactions provide the structural background of this structural feature. In MAP2c, such tertiary interactions are absent, which points to the importance of local structural constraints. In fact, urea and temperature dependence of the CD spectrum of its PRR reveals the presence of the extended and rather stiff polyproline II helix conformation that keeps the interaction site exposed. These data suggest that functionally significant residual structure exists in both of these IUPs. This structure, manifest as either transient local and/or global organization, ensures the spatial exposure of short contact segments on the surface. Pertinent data from other IUPs suggest that the presence of such recognition motifs may be a general feature of disordered proteins. To emphasize the possible importance of this structural trait, we propose that these motifs be called primary contact sites in IUPs.

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

“…1b). It has been observed that many IDPs exhibit regions of residual structure, which are believed to function as primary interaction sites (14). In support of this hypothesis, our study indicates that two of the functional secondary structural elements of PDE␥ are partially formed in the uncomplexed protein.…”

“…A surge of recent evidence has established that both denatured proteins and IDPs can contain residual structure and even adopt compact folds in solution (10)(11)(12)(13). The residual structure of an IDP is believed to play a role in initiating the folding process, in protein-protein interactions, or in preventing aggregation (10,14,15). Despite the great challenge to structure determination imposed by the dynamic properties of an IDP, recently developed NMR methods, such as paramagnetic relaxation enhancement (PRE) (16) and residual dipolar coupling (RDC) (17), have enabled detailed elucidation of their transient conformations.…”

Calcium‐induced tripartite binding of intrinsically disordered calpastatin to its cognate enzyme, calpain

“…The 15 N-and 15 N-13 C-labeled protein was expressed similarly. The protein was initially purified as described previously [5], and then on a C-18 HPLC column: final purity >98%.…”

Micelle-Induced Folding of Spinach Thylakoid Soluble Phosphoprotein of 9 kDa and Its Functional Implications,

“…Despite their lack of a globular structure, intrinsically disordered proteins play key roles in signal transduction and other critical cellular processes (59)(60)(61). Biochemical and biophysical characterizations of these unstructured proteins have revealed that residual structure, including permanent or transient secondary structural elements, can be present in these proteins and function as protein recognition motifs (62). Stabilization of the residual structure is often coupled with other activities: some unstructured proteins and peptides become folded upon binding to their biological targets (60,63,64); others are unfolded under aqueous conditions but fold in the presence of lipids or model membranes (65)(66)(67).…”

“…Thermodynamically, the induced-folding process is always accompanied by significant entropy-enthalpy compensation. It is believed that the physiological importance of the residual structure is to facilitate molecular recognition during protein-protein interaction (62), and the induced folding allows fine control over binding affinity (63). For TSP9, it is also possible that the micelle-induced folding controls its functional specificity in the thylakoid membrane.…”