A de novo designed protein protein interface

Abstract

As an approach to both explore the physical/chemical parameters that drive molecular self-assembly and to generate novel protein oligomers, we have developed a procedure to generate protein dimers from monomeric proteins using computational protein docking and amino acid sequence design. A fast Fourier transform-based docking algorithm was used to generate a model for a dimeric version of the 56-amino-acid beta1 domain of streptococcal protein G. Computational amino acid sequence design of 24 residues at the dimer interface resulted in a heterodimer comprised of 12-fold and eightfold variants of the wild-type protein. The designed proteins were expressed, purified, and characterized using analytical ultracentrifugation and heteronuclear NMR techniques. Although the measured dissociation constant was modest ( approximately 300 microM), 2D-[(1)H,(15)N]-HSQC NMR spectra of one of the designed proteins in the absence and presence of its binding partner showed clear evidence of specific dimer formation.

Figure 1.

(A) Ribbon representation of the GB1 monomer structure. (B) Tube representation of the docked GB1 dimer model; (left) GB1^A; (right) GB1^B. (C) Surface representation of the docked GB1 dimer model showing surface complementarity of the Cβ-truncated monomers used in the docking calculation; (left) GB1^A; (right) GB1^B.

Figure 2.

[¹⁵N, ¹H] HSQC spectra of uniformly enriched ¹⁵N-GB1^A alone (in red) and in the presence of equimolar quantities of unlabeled GB1^B (in black). Example ¹⁵N-monomer-A peaks that are nonobservable or exhibit chemical shift perturbations upon complex formation are labeled blue.

Figure 3.

(A) Chemical shift perturbations mapped to the surface of GB1^A in the docked model orientation together with GB1^B shown as a gray backbone worm on which the interfacial side chains are colored red. (Dark blue) Residues with nonobservable [¹⁵N, ¹H] HSQC peaks; (lighter blue) those that exhibit chemical shift changes. (B) 180° rotation of A.