GroEL/S substrate specificity based on substrate unfolding propensity

Abstract

Phage P22 wild-type (WT) coat protein does not require GroEL/S to fold but temperature-sensitive-folding (tsf ) coat proteins need the chaperone complex for correct folding. WT coat protein and all variants absolutely require P22 scaffolding protein, an assembly chaperone, to assemble into precursor structures termed procapsids. Previously, we showed that a global suppressor (su) substitution, T166I, which rescues several tsf coat protein variants, functioned by inducing GroEL/S. This led to an increased formation of tsf :T166I coat protein:GroEL complexes compared with the tsf parents. The increased concentration of complexes resulted in more assembly-competent coat proteins because of a shift in the chaperone-driven kinetic partitioning between aggregation-prone intermediates toward correct folding and assembly. We have now investigated the folding and assembly of coat protein variants that carry a different global su substitution, F170L. By monitoring levels of phage production in the presence of a dysfunctional GroEL we found that tsf :F170L proteins demonstrate a less stringent requirement for GroEL. Tsf :F170L proteins also did not cause induction of the chaperones. Circular dichroism and tryptophan fluorescence indicate that the native state of the tsf : F170L coat proteins is restored to WT-like values. In addition, native acrylamide gel electrophoresis shows a stabilized native state for tsf :F170L coat proteins. The F170L su substitution also increases procapsid production compared with their tsf parents. We propose that the F170L su substitution has a decreased requirement for the chaperones GroEL and GroES as a result of restoring the tsf coat proteins to a WT-like state. Our data also suggest that GroEL/S can be induced by increasing the population of unfolding intermediates.

Highly Specific Salt Bridges Govern Bacteriophage P22 Icosahedral Capsid Assembly: Identification of the Site in Coat Protein Responsible for Interaction with Scaffolding Protein

“…Although WT coat protein does not require chaperonins to fold in vivo, temperature-sensitive-folding coat mutants can be rescued by overexpression of the GroES/L proteins resulting in viable progeny phage (33)(34)(35). If the E5A and D14A coat mutants cause a folding defect, coexpression of these mutants with extra GroES/L might rescue the assembly of infectious phages, and plating efficiencies would be expected to be similar to that of WT phage.…”

Nature׳s favorite building block: Deciphering folding and capsid assembly of proteins with the HK97-fold

“…Likewise, P22 scaffolding protein has been shown to exhibit chaperone-like activity for coat protein variants that have folding defects. Two global suppressors ( su , D163G and T166I) of temperature-sensitive-folding ( tsf ) mutations in P22 coat protein rescue folding via enhanced interaction with scaffolding protein (Parent et al, 2004; Parent and Teschke, 2007). Thus, both HK97’s Δ-domain and P22’s scaffolding protein act as folding chaperones of their coat proteins by promoting assembly to a more stable state by partitioning the precursors to assembled particles before aggregation can occur.…”

‘Let the phage do the work’: Using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants

“…Three global su substitutions (D163G, T166I, F170L) were repeatedly and independently isolated from different gene 5 tsf parents, scattered throughout the gene. The tsf:su coat proteins demonstrate both stabilized native and intermediate states as compared to their tsf parents, so that these proteins remain GroEL substrates even though their folding is less defective (Doyle et al, 2004; Parent and Teschke, 2007). The three global su substitutions are located in the β-hinge, which implicates this region as important for folding (Figure 4d).…”

“…For example, T166I and D163G both fix the original tsf defects by a shared mechanism. However, F170L corrects tsf protein folding defects by a second mechanism (Aramli and Teschke, 2001; Doyle et al, 2004; Parent and Teschke, 2007). …”

“…GroEL/S is unable to rescue the folding of the tsf :T166I coat proteins when there are decreased levels of intracellular scaffolding protein. The D163G substitution appears to function by the same mechanism as T166I in vivo (Parent and Teschke, 2007). …”