Alanine Scan of Core Positions in Ubiquitin Reveals Links between Dynamics, Stability, and Function

Abstract

Mutations at solvent inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. The two null mutants (I30A and L43A) were both less stable to temperature-induced unfolding in vitro than wild-type, but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to wild-type. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high molecular weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high molecular weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation.

Systematic Exploration of Ubiquitin Sequence, E1 Activation Efficiency, and Experimental Fitness in Yeast

“…Of the 16 core positions, 13 have aliphatic side chains in wild-type ubiquitin (six Leu, four Ile, and three Val) that form a hydrophobic cluster known to be a driving force for stabilizing native protein structures 42; 43 . Consistent with observations that the protein folding stability of wild-type ubiquitin is far greater than required for yeast growth 27; 44 , we observed that modest substitutions to other aliphatic side chains were generally well tolerated for activation by E1 (Supplementary Fig. S3b).…”

Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting

“…While these caveats are a concern, the SUB328 experimental system has been successfully used for many years to assess the effect of Ub mutations in yeast (Roscoe et al, 2013;SloperMould et al, 2001;Lee et al, 2014). Furthermore, we have only interpreted mutants that cause large defects, such as the biologically sensible fitness defects of mutations at known important residues such as Leu8 or Lys48 and residues with fitness values that vary between conditions such as His68.…”

Viewing Protein Fitness Landscapes Through a Next-Gen Lens

“…Some of the most fundamental observations of protein conformational dynamics and their contributions to function come from NMR analyses of enzymes where highly specific protein motions can be required for efficient catalysis (Eisenmesser et al 2002;Villali and Kern 2010). Recent systematic analyses of mutations in proteins or regions of proteins involved in protein-protein interactions (Jiang et al 2013;Lee et al 2014; indicate that binding functions can also be very sensitive to protein conformational changes less severe than complete unfolding. This inference comes largely from observations that many amino acid substitutions at solvent inaccessible positions in these binding proteins cause strong functional defects without causing protein unfolding.…”

“…In addition, ubiquitin primarily acts through protein-protein binding interactions. Despite these features, many mutations in the core of ubiquitin that cause subtle alterations to the conformational properties of ubiquitin have large impacts on its binding functions (Phillips et al 2013;Lee et al 2014;. Further studies on additional proteins should provide valuable insights into the generality of these observations for binding proteins and the nature of biophysical constraints in these systems.…”