IndraLab

"The data were subsequently group fit with a common exchange rate for residues that satisfy χ 2 group /χ 2 invididual < 2.0. xref Group fitting yielded k ex = 4954 ± 82 s −1 for p-DUBA ( xref ). np-DUBA undergoes a similar conformational exchange process with the exchange rate of 5750 ± 110 s −1 ( xref )."

"A network of dynamic residues underlies the synchronized motions observed in both p-DUBA and np-DUBA and explains the similarity in the global conformational dynamics in these two forms."

"In the enzyme-substrate complex formed from np-DUBA, the α1 helix either cannot adopt a closed conformation or fluctuates rapidly between open and closed conformations."

"Motions in the apo state of enzymes can facilitate substrate binding by enabling transitions into to a binding competent state xref , xref and can also regulate the catalytic cycle by allowing access to conformational states conducive for catalytic turnover. xref – xref The low activity of np-DUBA likely results from its inability to form a highly productive enzyme-substrate complex rather than low binding affinity to substrates because only 3-fold enhancement in the affinity to the fluorogenic ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) substrate was observed upon phosphorylation, whereas the enhancement in k cat is 300-fold. xref We can speculate that an important feature of the productive enzyme-substrate complex is that the α1 helix can adopt a closed conformation, as defined by the crystal structure of p-DUBA in the ubiquitin-bound state. xref The only conformer in np-DUBA lies between the a and b conformers in terms of structural characteristics according to the relative positions of cross-peaks. xref This observation indicates that at least in the free state, the α1 helix in np-DUBA adopts a more open conformation than the a conformer in p-DUBA."

"Broadening of NMR spectral lines and the resulting low signal intensities are signatures of conformational exchanges on the μs-ms scales. xref Among ~ 120 cross-peaks detectable in the 15 N TROSY spectra of p-DUBA, many display broad 1 H lines, whereas the 15 N transverse relaxation rates of majority of the resonances are consistent with the molecular weight. xref , xref TROSY spectra of nonphosphorylated DUBA (np-DUBA) display similar features."

"The higher R 2,eff could result from motions that are too fast to yield relaxation dispersion and/or higher solvent exchange rate in np-DUBA."

"E200 is spatially close to the α1-α2 loop and similarly show decreased R ex in the p-DUBA compared to np-DUBA."

"Only one residue, E200, displays more than 2-fold difference in the R ex between p-DUBA and np-DUBA."

"In regions outside the N-terminal segment, p-DUBA and np-DUBA are highly similar ( xref )."

"Differences in the dynamics of p-DUBA and np-DUBA."

"Despite the overall similarity in the global conformational process between p-DUBA and np-DUBA, the α1 helix and neighboring regions display detectable differences."

"Most notably, S177 in np-DUBA shows a flat dispersion profile but elevated R 2,eff compared to the b conformer in p-DUBA ( xref )."

"The distinct motions of the α1 helix and the neighboring loops in p-DUBA and np-DUBA, especially the segment around S177, may explain the large difference in activity."

"The backbone assignments of p-DUBA and np-DUBA are available in the Biological Magnetic Resonance Bank (BMRB) under accession codes 27495 and 27494, respectively."

"In this work, we characterized the conformational dynamics of DUBA on the μs-ms time scales by 1 H CPMG experiments, which allowed us to discern the differences in the dynamic properties between p-DUBA and np-DUBA and those between the two conformers in p-DUBA."

"In np-DUBA, S177 displays a flat relaxation dispersion profile but elevated R 2,eff , which may result from motions too fast to be detected by the CPMG experiments."

"Based on this observation, we performed constant-time amide 1 H Carr-Purcell-Meiboom-Gill (CPMG) experiments xref to characterize the conformational dynamics of both p-DUBA and np-DUBA."