Conversion of monomeric protein L to an obligate dimer by computational protein design

Abstract

CorrectionsBIOCHEMISTRY. For the article ''Differential effects of a centrally acting fatty acid synthase inhibitor in lean and obese mice,'' by Monica V. Kumar, Teruhiko Shimokawa, Tim R. Nagy, and M. Daniel Lane, which appeared in number 4, February 19, 2002, of Proc. Natl. Acad. Sci. USA (99, 1921-1925, the authors note the following. ''Under a licensing agreement between FASgen, Inc., and The Johns Hopkins University, Dr. Lane is entitled to a share of royalty received by the University on sales of products that embody the technology described in this article. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict of interest policies. ' (98, 10687-10691; First Published August 28, 2001; 10.1073͞pnas.181354398), the authors note the following. In the Introduction and Discussion of our paper, we failed to reference a recent article by Rousseau et al. (1), which demonstrated that single point mutations can significantly perturb the equilibrium between monomeric and domain-swapped dimeric p13suc1. Rational methods were used to redesign p13suc1 from a fully monomeric protein (dissociation constant of Ϸ900 mM) to a fully dimeric protein (dissociation constant of Ϸ100 nM). Protein L consists of a single ␣-helix packed on a four-stranded ␤-sheet formed by two symmetrically opposed ␤-hairpins. We use a computer-based protein design procedure to stabilize a domainswapped dimer of protein L in which the second ␤-turn straightens and the C-terminal strand inserts into the ␤-sheet of the partner. The designed obligate dimer contains three mutations (A52V, N53P, and G55A) and has a dissociation constant of Ϸ700 pM, which is comparable to the dissociation constant of many naturally occurring protein dimers. The structure of the dimer has been determined by x-ray crystallography and is close to the in silico model.I n domain swapping, one structural element of a protein breaks its noncovalent bonds with the rest of the protein and reforms them with an identical partner (1, 2). The result is an intertwined dimer or a higher-order oligomer. All of the interactions that stabilize the monomer are present in the oligomer except for those in the hinge region that connects the swapped domain with the rest of its chain. Often the hinge region forms a loop or turn in the monomer, and just a few mutations to this region of the protein will induce formation of the domain-swapped oligomer. Several studies have shown that shortening the hinge region often stabilizes the domainswapped variant (3-7). In these cases it appears that the shorter loop prevents the chain from bending back and reinserting into the monomer, but the chain can easily continue forward into the partner of the domain-swapped structure. Another scenario involves leaving the loop length unchanged, but making amino acid substitutions that favor one hinge conformation over the other.The 62-residue IgG-binding domain of protein L is monomeric and consists of a single ␣-helix packed on a fourstranded ␤-sheet f...

Get5 Carboxyl-terminal Domain Is a Novel Dimerization Motif That Tethers an Extended Get4/Get5 Complex

“…Engineering dimerization into proteins is an important goal (49,50). Much of this type of work focuses on the use of coiledcoils in the form of leucine zippers (51).…”

Using Molecular Dynamics Simulations as an Aid in the Prediction of Domain Swapping of Computationally Designed Protein Variants

“…Studies have shown that mutating these critical residues can significantly affect domain-swapping tendency [26,27].…”

“…Given that domain-swapped dimer structures are usually not explicitly modeled in CPD [27]. Similarly, while attempting to design a homodimer from a 51 amino acid fragment of the monomeric protein engrailed homeodomain (ENH), we generated a high affinity dimer, ENH_DsD (Table S1), with a K d of ~40 nM that also proved to be domain-swapped when examined by X-ray crystallography.…”

“…We also investigated variants of the B1 domain of protein L from Peptostreptococcus magnus generated by Baker and coworkers [26,27] and variants of the B1 domain of streptococcal protein G generated by Gronenborn and coworkers [32,33]. O'Neill et al showed that the point mutant G55A of protein L is a weak domain-swapped dimer with a K d of ~30 μM [26].…”

“…used CPD to identify mutations that stabilize the domain-swapped dimer structure while destabilizing the monomer structure, creating a triple mutant (A52V/N53P/G55A) obligate dimer with a K d of ~700 pM [27]. The proline substitution at position 53 is not compatible with the structure of the monomer and is expected, at a minimum, to alter the local structure.…”