Positive selection dictates the choice between kinetic and thermodynamic protein folding and stability in subtilases.
Subtilisin E (SbtE) is a member of the ubiquitous superfamily of serine proteases called subtilases and serves as a model for understanding propeptide-mediated protein folding mechanisms. Unlike most proteins that adopt thermodynamically stable conformations, the native state of SbtE is trapped into a kinetically stable conformation. While kinetic stability offers distinct functional advantages to the native state, the constraints that dictate the selection between kinetic and thermodynamic folding and stability remain unknown. Using highly conserved subtilases, we demonstrate that adaptive evolution of sequence dictates selection of folding pathways. Intracellular and extracellular serine proteases (ISPs and ESPs, respectively) constitute two subfamilies within the family of subtilases that have highly conserved sequences, structures, and catalytic activities. Our studies on the folding pathways of subtilisin E (SbtE), an ESP, and its homologue intracellular serine protease 1 (ISP1), an ISP, show that although topology, contact order, and hydrophobicity that drive protein folding reactions are conserved, ISP1 and SbtE fold through significantly different pathways and kinetics. While SbtE absolutely requires the propeptide to fold into a kinetically trapped conformer, ISP1 folds to a thermodynamically stable state more than 1 million times faster and independent of a propeptide. Furthermore, kinetics establish that ISP1 and SbtE fold through different intermediate states. An evolutionary analysis of folding constraints in subtilases suggests that observed differences in folding pathways may be mediated through positive selection of specific residues that map mostly onto the protein surface. Together, our results demonstrate that closely related subtilases can fold through distinct pathways and mechanisms, and suggest that fine sequence details can dictate the choice between kinetic and thermodynamic folding and stability.
Subbian
Ezhilkani
E
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, USA.
Yabuta
Yukihiro
Y
Shinde
Ujwal
U
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D019143:Evolution, Molecular; D005110:Extracellular Space / Q000201:enzymology; D007424:Intracellular Fluid / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D020860:Subtilisin / Q000737:chemistry* / Q000378:metabolism*; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D004792:Enzyme Precursors; D012697:Serine Endopeptidases; D013381:Subtilisins; D020860:Subtilisin