The Native-like Interactions between SNase121 and SNase(111−143) Fragments Induce the Recovery of Their Native-like Structures and the Ability to Degrade DNA

Abstract

The interactions necessary for stabilizing the folding of the N-terminal large beta-subdomain and the C-terminal small alpha-subdomain of staphylococcal nuclease (SNase) were investigated by an approach of fragment complementation. Two SNase fragments, namely, SNase121 and SNase(111-143) containing 1-121 and 111-143 residues, respectively, of native SNase, were used in this study since the sequences of the two fragments correspond to that of the beta- and alpha-subdomains of SNase. SNase121 is a largely unfolded fragment whereas SNase(111-143) is a structureless fragment. The recognition process and efficiency of complementation of SNase121 and SNase(111-143) fragments were studied by NMR and various biochemical and biophysical methods. SNase121 and SNase(111-143) can recognize each other and recover their native conformations on binding, restoring the active site and the ability to degrade DNA. The SNase121:SNase(111-143) complex showed a nuclease activity up to 30% that of native SNase. The final rigid structures of SNase121 and SNase(111-143) fragments having the folded native-like beta-subdomain and alpha-subdomain structures of SNase, respectively, in the complex form simultaneously with the complex stabilization. Studies with the mutant SNase121 and SNase(111-143) fragments reveal that the sequence elements which are essential for recognition and efficient complementation of the two fragments are also necessary for recovering the native-like interactions at the binding interface between them. The interfragment interactions that induce the structural complementation of SNase121 and SNase(111-143) likely reflect the tertiary interactions necessary to stabilize the folding of both beta- and alpha-subdomains in the native SNase.

Importance of the C-Terminal Loop L137−S141 for the Folding and Folding Stability of Staphylococcal Nuclease,

“…Presumably, the C-terminal loop L137−S141 may have some inherent tertiary interactions with the helix, which contributes to the folding of helix α3. In native SNase, residues L137 and W140 in the loop L137−S141 are involved in the hydrophobic interactions with a long hydrophobic side chain (C β−ζ ) of an amphiphilic residue K133 in the helix α3 ,, . Besides, the NH of L137 forms a hydrogen bond with CO of A132 .…”

“…It seems the C-terminal loop L137−S141 may play a role of the C-terminal capping of helix α3 in SNase, since the loop L137−S141 has a helical-turn conformation for forming such hydrophobic C-capping interactions. In fact, the hydrophobic C-capping interactions for folding of helix α3 are a component part of the hydrophobic packing interactions of the pyrrole ring of W140 with the hydrophobic side chains of surrounding residues G107, L108, A109, and amphiphilic K110 from the β-subdomain and A132, L137, I139, and amphiphilic K133 from the α-subdomain , , which serve as an anchoring force for specific association between the α- and β-subdomains of SNase . Together, these hydrophobic interactions make the local structural region surrounding W140 a small C-terminal hydrophobic core in native SNase.…”

“…SNase is a widely used model system for the study of protein folding, which is composed of two subdomains, a large N-terminal β-subdomain and a small C-terminal α-subdomain. The N-terminal β-subdomain is constructed by a “β-barrel” core and two helices α1 (A58−A69) and α2 (V99−Q106) as well as the p-loop (pdTp-binding loop, D77−L89) and Ω-loop (P42−P56), whereas the C-terminal α-subdomain contains mainly a helix α3 (E122−K136) and the subsequent helical turn L137−S141 and the N- and C-terminal loops of the subdomain , . A mixture of the N-terminal β-subdomain fragment and the C-terminal α-subdomain fragment can form a noncovalent complex through a folding-on-binding event, which generates a tertiary structure nearly identical to the structure of native SNase.…”

“…A mixture of the N-terminal β-subdomain fragment and the C-terminal α-subdomain fragment can form a noncovalent complex through a folding-on-binding event, which generates a tertiary structure nearly identical to the structure of native SNase. For this, the crucial elements in the surface region of the binding interface between two subdomains are required, which establish the interactions necessary for stabilizing the native-like folding of both fragments . The C-terminal amino acids 137−149 of SNase were proposed to contain critical information for the native folding of SNase , .…”