Single-Site Mutations Induce 3D Domain Swapping in the B1 Domain of Protein L from Peptostreptococcus magnus

O’Neill, Jason; Kim, David E.; Johnsen, Keyji; Baker, David; Zhang, Kam Y. J.

doi:10.1016/s0969-2126(01)00667-0

Cited by 51 publications

(47 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similar observations about the role of strained hinge loops in domain swapping have since been made in the B1 domain of protein L (17). The monomer-dimer equilibrium of suc1 can be monitored by analytical gel filtration chromatography, and the K d obtained for wild type was 1.8 mM; at protein concentrations in the 1-5 M range, suc1 is Ͼ99% monomeric at equilibrium (14).…”

Section: Resultssupporting

confidence: 65%

Intermediates Control Domain Swapping during Folding of p13

Rousseau

Schymkowitz

Wilkinson

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The 13-kDa protein p13 suc1 has two folded states, a monomer and a structurally similar domain-swapped dimer formed by exchange of a ␤-strand. The refolding reaction of p13 suc1 is multiphasic, and in this paper we analyze the kinetics as a function of denaturant and protein concentration and compare the behavior of wild type and a set of mutants previously designed with dimerization propensities that span 9 orders of magnitude. We show that the folding reactions of wild type and all mutants produce the monomer predominantly despite their very different equilibrium behavior. However, the addition of low concentrations of denaturant in the refolding buffer leads to thermodynamic control of the folding reaction with products that correspond to the wild type and mutant equilibrium dimerization propensities. We present evidence that the kinetic control in the absence of urea arises because of the population of the folding intermediates. Intermediates are usually considered to be detrimental to folding because they slow down the reaction; however, our work shows that intermediates buffer the monomeric folding pathway against the effect of mutations that favor the nonfunctional, dimeric state at equilibrium. p13 suc1 (referred to subsequently as suc1), 1 a member of the Cks family of cell cycle regulatory proteins, has two native states: a monomer (1) and a domain-swapped dimer formed by exchange of a central strand, ␤4, of the ␤-sheet (2, 3) ( Fig. 1). Each subunit of the dimer is highly superimposable on the monomer except for the so-called "hinge loop" that connects the swapping strand to the rest of the structure. The hinge loop forms a ␤-hairpin in the monomer and has an extended conformation in the dimer. In this work we present an global model of the folding and interconversion of monomeric and dimeric suc1. The description illustrates how domain swapping, by exploiting mostly native interactions, creates ambiguity in protein folding that can lead to misfolding and aggregation. Unexpectedly, however, we find that intermediates can protect the folding reaction and ensure that it leads to the correct oligomeric native product.Recent theoretical and experimental work on folding of small proteins has shown that the native state topology defines the general features of protein folding pathways, and that sequence specifities modulate it to some extent (4 -6). This view raises two broad questions: 1) How do protein sequences favor native-like interactions at each stage of the folding process and avoid misfolding? Do proteins possess gatekeeper residues that restrict the conformational space to native regions (7), or is the overall energy bias in favor of native interactions rather than non-native ones sufficient to guide folding? 2) Even if only native interactions are favored, it is still possible to make native interactions with residues from another polypeptide chain rather than intramolecularly by the process of domain swapping? How do proteins avoid these undesirable interactions and ensure folding to the correct...

show abstract

Section: Resultssupporting

confidence: 65%

Intermediates Control Domain Swapping during Folding of p13

Rousseau

Schymkowitz

Wilkinson

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…(the bases for the VPA model), which represents the asymmetric unit (10). Residues A52 and D53 were truncated to glycine in the G55A model.…”

Section: Figmentioning

confidence: 99%

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

Kuhlman

O’Neill

Kim

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

“…Dimers and higher-order oligomers can evolve in a single step by this mechanism. In fact, single mutations induce 3D domain swap dimers in a relative of protein G (13), and one set of five conservative mutations leads to a tetramer formed through the exchange of ␤-strands (14). Taken together, the data suggest that the N and C termini of folded proteins provide evolution with a variety of opportunities for formation of new structuresrepositioning and rearrangement of the ends, oligomerization by 3D domain swapping, and sites for accretion of additional amino acids by nonhomologous recombination.…”

mentioning

confidence: 99%

One sequence plus one mutation equals two folds

Shortle

2009

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Single-Site Mutations Induce 3D Domain Swapping in the B1 Domain of Protein L from Peptostreptococcus magnus

Cited by 51 publications

References 45 publications

Intermediates Control Domain Swapping during Folding of p13

Intermediates Control Domain Swapping during Folding of p13

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

One sequence plus one mutation equals two folds

Contact Info

Product

Resources

About