A minimal sequence code for switching protein structure and function

Abstract

We present here a structural and mechanistic description of how a protein changes its fold and function, mutation by mutation. Our approach was to create 2 proteins that (i) are stably folded into 2 different folds, (ii) have 2 different functions, and (iii) are very similar in sequence. In this simplified sequence space we explore the mutational path from one fold to another. We show that an IgG-binding, 4␤؉␣ fold can be transformed into an albumin-binding, 3-␣ fold via a mutational pathway in which neither function nor native structure is completely lost. The stabilities of all mutants along the pathway are evaluated, key high-resolution structures are determined by NMR, and an explanation of the switching mechanism is provided. We show that the conformational switch from 4␤؉␣ to 3-␣ structure can occur via a single amino acid substitution. On one side of the switch point, the 4␤؉␣ fold is >90% populated (pH 7.2, 20°C). A single mutation switches the conformation to the 3-␣ fold, which is >90% populated (pH 7.2, 20°C). We further show that a bifunctional protein exists at the switch point with affinity for both IgG and albumin.evolution ͉ NMR ͉ protein design ͉ protein folding

GA/GB Fold switching may modulate fatty acid transfer from human serum albumin to bacteria

“…Bacterial GA modules have recently attracted the interests of scientists in the protein fold field because of a seminal study involving the two binding domains of protein G (GA and GB) (16). GA and GB are small domains of similar length which display different folds, the GA domain being characterized by an all-a fold, whereas the GB domain displays a 4b1a fold (16).…”

“…GA and GB are small domains of similar length which display different folds, the GA domain being characterized by an all-a fold, whereas the GB domain displays a 4b1a fold (16). Interestingly, through a series of point mutations which increase the sequence identity between the two domains, variants which share 98% sequence identity (namely, GA98 and GB98) but retain a different fold (all-a in the case of GA98 and 4b1a in the case of GB98) have been generated (16). Remarkably, no unique amino acid appears to dictate for the GA rather than the GB topology, and a series of variants differing in a single position could be designed for the twofolds (17).…”

Hydrophobic interactions in the formation of secondary structures in small peptides

“…Secondary structure formation is context dependent [14,65]. It follows that a secondary structure that is favored in the protein core might not be favored under denaturing conditions.…”

“…In the case of the arc repressor homodimer, interchanging the positions of a hydrophobic and a polar residue turns a two-strand β sheet into a pair of helices [13]. Such hydrophobicity-related conformational switches [14] have important ramifications for molecular evolution [15]. Indeed, in de novo protein design, an important step is to choose a hydrophobic-polar pattern that packs nonpolar residues in secondary structures well within a core [16].…”

Quantitative theory of hydrophobic effect as a driving force of protein structure

“…It is possible that a protein's native fold might be well-structured but that it could exhibit strong sensitivity to small changes in its sequence. For example, in a recent study, Alexander et al 24 demonstrated that it is possible to design a version of the streptococcal protein G such that a single point mutation (L45Y) leads to switching from 3a to 4b 1 a fold. Furthermore, they obtained high-resolution NMR structures of two proteins (2KDL, 2KDM) different by three mutations (L20A, I30F, L45Y).…”