Ubiquitin's Conformational Heterogeneity as Discerned by Nuclear Magnetic Resonance Spectroscopy

Abstract

Visualizing a protein's molecular motions has been a long standing topic of research in the biophysics community. Largely this has been done by exploiting nuclear magnetic resonance spectroscopy (NMR), and arguably no protein's molecular motions have been better characterized by NMR than that of ubiquitin (Ub), a 76 amino acid polypeptide essential in ubiquitination-a key regulatory system within cells. Herein, we discuss ubiquitin's conformational plasticity as visualized, at atomic resolution, by more than 35 years of NMR work. In our discussions we point out the differences between data acquired in vitro, ex vivo, as well as in vivo and stress the need to investigate Ub's conformational plasticity in more biologically representative backgrounds.

Keywords: Conformational heterogeneity; NMR; Post-translational modifications; Protein dynamics; in-cell NMR.

Figure 1

Insight into Ub's structure and into the amplitudes and associated timescales of protein motions. Panel A. Ub's structure as revealed by NMR with its seven internal lysine moieties highlighted (K6, K11, K27, K29, K33, K48, K63) in maroon (PDB ID: 1D3Z). Panel B. Amplitudes of protein motions with associated timescales and their detection by various solution‐state NMR experiments–NOE–Nuclear Overhauser effect, CEST – chemical exchange saturation transfer.

Figure 2

Free monomeric Ub's conformational heterogeneity revealed by high‐pressure NMR. Superposition of low/high‐pressure conformers −3 bar (white) and 30 bar (cyan) readily reveals cavity expansion with concomitant stark conformational changes (see inset) for E24 and D52 which act as a “hinge”. PDB ID 1V80 and 1V81.

Figure 3

Overview of free monomeric Ub's dynamic segments as revealed by solution‐state NMR. Panel A. Depiction of Cα root mean square fluctuations in RDC‐derived structures‐plotted on PDB 1UBQ and as a graph–data were extracted from Ref. Cα residues with RMSFs exceeding >0.5 Å are highlighted in red on the sequence with α‐helices and β‐strands being denoted in black and violet, respectively. Panel B. Cα RMSD for residues undergoing ps to ns motions as revealed by modeling accounting for distance and relaxation constraints plotted on PDB ID 1UBQ–extracted from Ref. Similarly, Cα residues with RMSDs>0.5 Å are highlighted in red with the same colouring scheme as above for secondary structure elements.

Figure 4

Free monomeric Ub's collective motions as revealed by solution‐state NMR. Panel A. Depiction of Ub's collective pincer motion that is centered on the β₁‐β₂ and β₄–α₂ loops causing expansion and contraction of the central cavity by displacement of α₁; arrows indicate direction of motion. Panel B. Depiction of Ub's collective peptidyl‐flip motion centered on D52 and G53. G53’s peptidyl flip alters the hydrogen bonding to E24 thus engendering a global allosteric switch and an inward rotating motion of the α‐helix; arrows depict direction of motion. Panel C/D. In the X‐ray crystallography structure (C) 1UBQ (white), E24’s sidechain can be seen pointing away from G53 whereas in the peptidyl flipped structure (D) 3ONS (cyan) E24 points towards G53 thereby facilitating a new hydrogen bonding pattern.

Figure 5

Ub's conformational heterogeneity. Panel A. Visualization of the loop‐in and loop‐out L8 conformers–1UBQ white and 2G45 cyan. Panel B. Overlay of the relaxed (white‐ 5XK4) and retracted (cyan‐ 5XK5) states of S65‐phosphorylated Ub as revealed by Dong et al. Panel C. Ub‐S65 phosphorylation induces significant allosteric changes in all β‐strands with shortening (β1) and lengthening (β2) being evident. These changes are concomitant with alterations in the hydrogen bonding pattern.

Figure 6

Overview of both free monomeric and conjugated Ub dynamics. Panel A. Summary of free monomeric Ub's conformational plasticity as determined by NMR studies (PDB ID:1UBQ for static structure and structural changes shown are extracted from PDB ID: 2K39). Panel B. Illustration of Ub‐chain dynamics; depicted is a linear Ub chain with cognate Ub's undergoing intrinsic structural fluctuations depicted in Panel A and that are then coupled with the chain's intrinsic motions. Panel C. Ub's sequence with α‐helix and β‐strand regions denoted in black and violet, respectively. Residues exhibiting ps to ms motions as per Ref. are denoted in red. Ubiquitination (Ub) sites are denoted in blue, SUMOylation (SUMO) sites in cyan, acetylation (Ac) in orange, carbamylation (CO₂) in maroon, phosphorylation (Ph) in green, ADP‐ribosylation (ADPR) in pink. Adapted from Ref. [1,72,143,144].