title: 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 (β/α)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.
  
  
  METHODS
  The coordinates of RBSX, V1A and V1L have been deposited in the PDB database under the accession numbers 4QCE, 4QCF, and 4QDM, respectively.
authors.affiliation: Department of Physics, Indian Institute of Science, Bangalore, India.
authors.forename: Pranjal
authors.initials: P
authors.lastname: Mahanta
authors.affiliation: Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Bhardwaj
authors.affiliation: Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
authors.forename: Krishan
authors.initials: K
authors.lastname: Kumar
authors.affiliation: Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
authors.forename: Vanga S
authors.initials: VS
authors.lastname: Reddy
authors.affiliation: Department of Physics, Indian Institute of Science, Bangalore, India.
authors.forename: Suryanarayanarao
authors.initials: S
authors.lastname: Ramakumar
mesh_terms: D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases; D014305:Triose-Phosphate Isomerase
keywords: glycosyl hydrolase family 10 (GH10) xylanase; residue interaction network; structure-thermostability relationship; terminal contact; triosephosphate isomerase (TIM) barrel