Ranjani Varadan

Diverse polyubiquitin interaction properties of ubiquitin-associated domains

The ubiquitin-associated (UBA) domain occurs frequently in proteins involved in ubiquitin-dependent signaling pathways. Although polyubiquitin chain binding is considered to be a defining feature of the UBA domain family, the generality of this property has not been established. Here we have surveyed the polyubiquitin interaction properties of 30 UBA domains, including 16 of 17 occurrences in budding yeast. The UBA domains sort into four classes that include linkage-selective polyubiquitin binders and domains that bind different chains (and monoubiquitin) in a nondiscriminatory manner; one notable class (∼30%) did not bind any ubiquitin ligand surveyed. The properties of a given UBA domain are conserved from yeast to mammals. Their functional relevance is further suggested by the ability of an ectopic UBA domain to alter the specificity of a deubiquitylating enzyme in a predictable manner. Conversely, non-UBA sequences can modulate the interaction properties of a UBA domain.

Structural properties of polyubiquitin chains in solution.

Because polyubiquitin chain structure modulates Ub-mediated signaling, knowledge of the physiological conformations of chain signals should provide insights into specific recognition. Here, we characterized the solution conformations of K48-linked Ub(2) and Ub(4) using a combination of NMR techniques, including chemical shift mapping of the interdomain interface, domain orientation measurements on the basis of 15N relaxation and residual dipolar couplings, and the solvent accessibility studies. Our data indicate a switch in the conformation of Ub(2), from open to closed, with increasing pH. The closed conformation features a well-defined interface that is related to, but distinguishable from, that observed in the Ub(2) crystal structure. This interface is dynamic in solution, such that important hydrophobic residues (L8, I44, V70) that are sequestered at the interface in the closed conformation may be accessible for direct interactions with recognition factors. Our results suggest that the distal two units of Ub(4), which is the minimum signal for efficient proteasomal degradation, may adopt the closed Ub(2) conformation.

Effects of cyclization on conformational dynamics and binding properties of Lys48-linked di-ubiquitin.

In solution, Lys48‐linked di‐ubiquitin exists in dynamic equilibrium between closed and open conformations. To understand the effect of interdomain motion in polyubiquitin chains on their ability to bind ligands, we cyclized di‐ubiquitin by cross‐linking the free C terminus of the proximal ubiquitin with the side chain of residue 48 in the distal ubiquitin, using a chemical cross‐linker, 1,6‐Hexane‐bis‐vinylsulfone. Our NMR studies confirm that the cyclization affects conformational dynamics in di‐ubiquitin by restricting opening of the interface and shifting the conformational equilibrium toward closed conformations. The cyclization, however, did not rigidly lock di‐ubiquitin in a single closed conformation: The chain undergoes slow exchange between at least two closed conformations, characterized by interdomain contacts involving the same hydrophobic patch residues (Leu8‐Ile44‐Val70) as in the uncyclized di‐ubiquitin. Lowering the pH changes the relative populations of these conformations, but in contrast with the uncyclized di‐ubiquitin, does not lead to opening of the interface. This restriction of domain motions inhibits direct access of protein molecules to the hydrophobic patch residues located at the very center of the interdomain interface in di‐ubiquitin, although the residual motions are sufficient to allow access of small molecules to the interface. This renders di‐ubiquitin unable to bind protein molecules (e.g., UBA2 domain) in the normal manner, and thus could interfere with Ub2 recognition by various downstream effectors. These results emphasize the importance of the opening/closing domain motions for the recognition and function of di‐ubiquitin and possibly longer polyubiquitin chains.

Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain

Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.