Residual Structure in the Denatured State of the Fast-Folding UBA(1) Domain from the Human DNA Excision Repair Protein HHR23A

Abstract

The structure of the first ubiquitin-associated domain from HHR23A, UBA(1), was determined by X-ray crystallography at a 1.60 Å resolution, and its stability, folding kinetics, and residual structure under denaturing conditions have been investigated. The concentration dependence of thermal denaturation and size-exclusion chromatography indicate that UBA(1) is monomeric. Guanidine hydrochloride (GdnHCl) denaturation experiments reveal that the unfolding free energy, ΔG u°′(H2O), of UBA(1) is 2.4 kcal mol–1. Stopped-flow folding kinetics indicates sub-millisecond folding with only proline isomerization phases detectable at 25 °C. The full folding kinetics are observable at 4 °C, yielding a folding rate constant, k f, in the absence of a denaturant of 13,000 s–1 and a Tanford β-value of 0.80, consistent with a compact transition state. Evaluation of the secondary structure via circular dichroism shows that the residual helical structure in the denatured state is replaced by polyproline II structure as the GdnHCl concentration increases. Analysis of NMR secondary chemical shifts for backbone 15NH, 13CO, and 13Cα atoms between 4 and 7 M GdnHCl shows three islands of residual helical secondary structure that align in sequence with the three native-state helices. Extrapolation of the NMR data to 0 M GdnHCl demonstrates that helical structure would populate to 17–33% in the denatured state under folding conditions. Comparison with NMR data for a peptide corresponding to helix 1 indicates that this helix is stabilized by transient tertiary interactions in the denatured state of UBA(1). The high helical content in the denatured state, which is enhanced by transient tertiary interactions, suggests a diffusion–collision folding mechanism.

High-Accuracy Prediction of Stabilizing Surface Mutations to the Three-Helix Bundle, UBA(1), with EmCAST

“…Most of the observed dihedral angles in UBA(1) fall within 1) is represented as its sequence of overlapping tetrads (x-axis). The observed Cα dihedral angles (τ, y-axis) of UBA(1) (pdb: 6W2H) 26 are shown on the plot as black or white open circles. The distribution of samples in our fragment database is rendered as heat (red = most populated, black, zero population).…”

“…Stabilizing helix 2 through Ncapping (E176T) or helix 3 through helix dipole optimization (H192E) yielded dramatic increases in the folding rates. These observations are consistent with the diffusion−collision model, 26,42 wherein the helices form early in the folding process and subsequently dock onto each other. E176T, while nearly identical to H192E in terms of its effect on stability, provides a notably larger acceleration of the folding process.…”

“…The pGEX-2T(TEV) plasmid containing the UBA(1) gene was used as a template for sitedirected mutagenesis. 26 Site-directed mutagenesis was carried out by using the QuikChange Lightning PCR-based mutagenesis kit (Agilent). Primers for mutagenesis were obtained from Invitrogen (Table S9).…”

AlphaFold2-RAVE: From sequence to Boltzmann ensemble

“…We present illustrative results using AlphaFold2-RAVE on proteins with unique challenges: the 1HZB CSP 11 for side-chain orientation predictions, ubiquitin binding protein UBA2 for disorder effects of missense mutations 4,12–14 , and SSTR2 GPCR for conformational diversity predictions 15 . For each, we show that AlphaFold2 fails, even with the reduced MSA trick from Ref.…”

AlphaFold2-RAVE: From Sequence to Boltzmann Ranking

“…We present illustrative results using AlphaFold2-RAVE on proteins with unique challenges: the 1HZB CSP for side-chain orientation predictions, ubiquitin binding protein UBA2 for disorder effects of missense mutations, ,− and SSTR2 GPCR for conformational diversity predictions . For each, we show that AlphaFold2 fails, even with the reduced MSA trick from ref .…”