Linkage-specific conformational ensembles of non-canonical polyubiquitin chains

Abstract

Polyubiquitination is a critical protein post-translational modification involved in a variety of processes in eukaryotic cells. The molecular basis for selective recognition of the polyubiquitin signals by cellular receptors is determined by the conformations polyubiquitin chains adopt; this has been demonstrated for K48- and K63-linked chains. Recent studies of the so-called non-canonical chains (linked via K6, K11, K27, K29, or K33) suggest they play important regulatory roles in growth, development, and immune system pathways, but biophysical studies are needed to elucidate the physical/structural basis of their interactions with receptors. A first step towards this goal is characterization of the conformations these chains adopt in solution. We assembled diubiquitins (Ub2) comprised of every lysine linkage. Using solution NMR measurements, small-angle neutron scattering (SANS), and in silico ensemble generation, we determined population-weighted conformational ensembles that shed light on the structure and dynamics of the non-canonical polyubiquitin chains. We found that polyubiquitin is conformationally heterogeneous, and each chain type exhibits unique conformational ensembles. For example, K6-Ub2 and K11-Ub2 (at physiological salt concentration) are in dynamic equilibrium between at least two conformers, where one exhibits a unique Ub/Ub interface, distinct from that observed in K48-Ub2 but similar to crystal structures of these chains. Conformers for K29-Ub2 and K33-Ub2 resemble recent crystal structures in the ligand-bound state. Remarkably, a number of diubiquitins adopt conformers similar to K48-Ub2 or K63-Ub2, suggesting potential overlap of biological function among different lysine linkages. These studies highlight the potential power of determining function from elucidation of conformational states.

Linkage via K27 Bestows Ubiquitin Chains with Unique Properties among Polyubiquitins

“…Therefore, we used CSPs in the distal Ub as indicators of noncovalent interactions between the Ub units in each Ub 2 . Our results suggest that, except for K6- and K48-Ub 2 , the noncovalent interdomain contacts in the rest of Ub 2 s are weak or transient, but nevertheless involve the hydrophobic surface patch (residues L8, I44, V70 (Beal et al, 1996)) of the distal Ub (Castañeda et al, 2016). Notably, the distal Ub in K27-Ub 2 exhibited the smallest CSPs of all the Ub 2 s studied.…”

“…We employed solution NMR spectroscopy to attain atom-specific information for each Ub 2 (Castañeda et al, 2016). 1 H- 15 N NMR spectra were collected separately for each Ub unit (uniformly 15 N-enriched) in K6, K11, K27, K29, K33, and K48-Ub 2 .…”

“…We used the sparse ensemble selection (SES) method (Berlin et al, 2013; Castañeda et al, 2016), to determine representative conformational ensembles for K27-Ub 2 from a starting set of 23000 sterically-allowed structures of the chain, generated in silico using SASSIE (Curtis et al, 2012) (Fig S6). …”

Investigating Structure and Dynamics of Proteins in Amorphous Phases Using Neutron Scattering

“…SasCalc (golden vector method) [35] allows the computation of neutron scattering profiles from structures and it can be freely accessed from a website [36]. Examples of this methodology are published in the literature [29], [34], [37], [38], [39], [40]. In the absence of crystallographic data, there are tools to generate molecular shapes that fit the experimental data, such as DAMMIF [41].…”

Top‐down analysis of novel synthetic branched proteins

“…In the present work, the replacement of an acidic residue with a basic residue at position 63 in Rub1 T72R does not modify the course of the conjugation reaction, to the extent that this is reflected in the attachment site for rubylation of ubiquitin. (The modifications at the structurally flexible carboxyl terminus of ubiquitin in these two examples prevent it from being activated by its E1 enzyme but do not impact the ubiquitin fold …”