Structural insights into N‐terminal to C‐terminal interactions and implications for thermostability of a (β/α)₈‐triosephosphate isomerase barrel enzyme

Abstract

Although several factors have been suggested to contribute to thermostability, the stabilization strategies used by proteins are still enigmatic. Studies on a recombinant xylanase from Bacilllus sp. NG-27 (RBSX), which has the ubiquitous (b/a) 8 -triosephosphate isomerase barrel fold, showed that just a single mutation, V1L, although not located in any secondary structural element, markedly enhanced the stability from 70°C to 75°C without loss of catalytic activity. Conversely, the V1A mutation at the same position decreased the stability of the enzyme from 70°C to 68°C. To gain structural insights into how a single extreme N-terminus mutation can markedly influence the thermostability of the enzyme, we determined the crystal structure of RBSX and the two mutants. On the basis of computational analysis of their crystal structures, including residue interaction networks, we established a link between N-terminal to C-terminal contacts and RBSX thermostability. Our study reveals that augmenting N-terminal to C-terminal noncovalent interactions is associated with enhancement of the stability of the enzyme. In addition, we discuss several lines of evidence supporting a connection between N-terminal to C-terminal noncovalent interactions and protein stability in different proteins. We propose that the strategy of mutations at the termini could be exploited with a view to modulate stability without compromising enzymatic activity, or in general, protein function in diverse folds where N and C termini are in close proximity. DatabaseThe coordinates of RBSX, V1A and V1L have been deposited in the PDB database under the accession numbers 4QCE, 4QCF, and 4QDM, respectively Abbreviations <n>, average number of nearest neighbors; Bc-Csp, cold shock protein from Bacillus caldolyticus; Bs-CspB, cold shock protein from Bacillus subtilis; BSX, xylanase from Bacilllus sp. NG-27; E, total number of edges or links; GH10, glycosyl hydrolase family 10; I s , interaction score; I smin , interaction score cut-off; LSCC, largest strongly connected component; N, total number of residues in the protein structure; PDB, Protein Data Bank; RBSX, recombinant xylanase from Bacilllus sp. NG-27; RIN, residue interaction network; RWC β CO, residue-wise C β contact order; SSE, secondary structural element; TIM, triosephosphate isomerase; T m , melting temperature.

Small Glycols Discover Cryptic Pockets on Proteins for Fragment-based Approaches

“…1B). It must be noted that this surface pocket was not observed in the native RBSX structure (PDB code 4QCE) [23]. Analysis of atoms lining the surface pocket in 5EFD (chain A) and their atom-wise solvent accessibilities (ASA (Å 2 )) revealed that this surface pocket is composed of both polar and apolar atoms (Table 2).…”

“…Indexing and integration of all the X-ray data sets was carried out using iMOSFLM [43] followed by scaling and merging with AIMLESS [44] program in CCP4 program suite [45]. Molecular replacement method using the program Phaser-MR [46] from the CCP4 program suite, was used for structure solution with native RBSX structure (PDB code 4QCE) [23] as the search model. Automated model rebuilding and completion of the molecular replacement solution was carried out using Phenix AutoBuild Wizard which uses RESOLVE, xtriage and phenix.refine to build an atomic model, refine it, and improve the same with iterative density modification, refinement, and model building [47].…”

Role of long-range aromatic clique and community in protein stability

“…An in silico analysis of a set of two-state folding proteins showed the presence of an N-C motif (N-terminal to C-terminal contacts) and suggested its possible role in initial protein folding, native state stability and final turnover [19]. In a recent study, we have provided experimental evidence through crystal structure analysis of different extreme N-terminus aliphatic mutants of RBSX where augmenting N-terminal to C-terminal interactions is associated with enhancement of the stability of the enzyme [11]. The present results are consistent with earlier findings and exemplify the importance of interactions between N-terminus and C-terminus specifically through aromatic interactions and provide a network perspective of aromatic interactions/cluster in modulating the stability of the enzyme.…”

“…RBSX is a monomeric GH10 xylanase having (β/α) 8 -TIM barrel fold [11]. The native structure of RBSX is composed of twelve α -helices, nine β -strands and five 3 10 -helices, as assigned by DSSP [12].…”

“…The availability of experimentally determined aromatic mutant structures has enabled to examine the local changes at the site of mutation. Evaluation of atomic packing as assessed with the RINERATOR module [13,14] shows that Phe4, Trp6 and Tyr343 in RBSX structure has a better noncovalent interaction score (I s ) [11] (noncovalent I s is proportional to strength of interaction) than Ala4, Ala6 and Ala343 in the F4A (PDB ID: 5EFF), W6A (PDB ID: 5EFD) and Y343A (PDB ID: 5EBA) mutant structures respectively (detail not shown).…”