Crystal structure of a streptococcal protein G domain bound to an Fab fragment

Derrick, Jeremy P.; Wigley, Dale B.

doi:10.1038/359752a0

Cited by 175 publications

(111 citation statements)

References 16 publications

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“…High-resolution structures were determined for G A 95 and G B 95 and provide insight into the structural (36) and GB77 in complex in IgG (from 1fcc.pdb) (37). The side chains of amino acids at the 13 positions of nonidentity are depicted as yellow sticks.…”

Section: Resultsmentioning

confidence: 99%

A minimal sequence code for switching protein structure and function

Alexander

He²,

Chen³

et al. 2009

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

232

330

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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

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Section: Resultsmentioning

confidence: 99%

A minimal sequence code for switching protein structure and function

Alexander

He²,

Chen³

et al. 2009

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

232

330

View full text Add to dashboard Cite

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“…For the formation of the disulfide-bridged dimer, the cysteine residue in the ext sequence of each monomer must be brought together into close proximity. This has been achieved by the tandem repeat of the Streptococcal protein G Fab binding domain (domain III) (11,15,16). The tandem repeats in this study have domains connected by G 4 S linker, which is known to have a very flexible conformation (17).…”

Section: Discussionmentioning

confidence: 99%

“…It has a serum albumin binding domain and three IgG binding domains. It has been identified that the third IgG binding domain (domain III) of Streptococcal protein G binds to the Fab fragment of IgG (11).…”

mentioning

confidence: 99%

Enhanced Formation of Disulfide-bridged Dimer (Fab-PE38)2 Utilizing Repeats of the Fab Binding Domain of Protein G

Lee

Park

Kweon

et al. 2010

Journal of Biological Chemistry

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Fab-PE38 used in this study is B3(Fab)-ext-PE38, and it is an antibody toxin that is made by fusing theAntibody toxins are made by fusing toxins to specific antibodies that selectively bind to cancer cells (1). (B3(Fab)-ext-PE38) 2 is the disulfide-bridged dimer of monomer B3(Fab)-ext-PE38 (2). We used the B3 monoclonal antibody as a model antibody, and it binds to a carbohydrate antigen found on many carcinomas (3, 4). PE38 is a 38-kDa derivative of Pseudomonas exotoxin (PE) 2 that catalyzes ADP-ribosylation of protein elongation factor 2 and inactivates it (5, 6). PE38 is a truncated form of the polypeptide that cannot bind to target cells by itself.B3(Fab)-ext-PE38 contains the light chain (V L -C L ) and the long chain in which the Fd chain (V H -C H1 ) is fused to PE38. The ext connects the Fd chain and PE38, producing the long Fd(V H -C H1 )-ext-PE38 chain. The Fab domain is formed by the formation of an interchain disulfide bond between the light chain and Fd chain. Two cysteines for this interchain disulfide bond are the one located at the end of C L domain of the light chain and the other one at the end of the C H1 domain of the long chain. The Fab toxin has two advantages over the single chain Fv(scFv) toxin in which two variable domains (V H and V L ) of antibody are connected with flexible linker. First, the refolding yield of Fab toxin is 10-fold higher than that of scFv toxin (7,8). Second, the stability of the Fab antibody toxin is higher than that of scFv toxin (9, 10).(B3(Fab)-ext-PE38) 2 has higher cytotoxicity than the monomeric B3(scFv)-PE38. Specifically, it has about 11-fold higher cytotoxicity in the CRL-1739 cell line than that of B3(scFv)-PE38 (2). The dimer is produced by the formation of a disulfide bond bridge between two monomers with the Cys residue located in the ext region in the Fd(V H -C H1 )-ext-PE38 chain. This disulfide bond is formed by the random collision of two Cys residues in two monomers. In our previous report, the refolding yield of the dimer was 0.06% (2). This is very low when compared with the yield of the monomer, which was ϳ10%.A dimeric antibody toxin is predicted to have several advantages over monomeric antibody toxins. The existence of two antigen binding domains should confer an increase in binding avidity. A dimeric antibody toxin will deliver two toxins at the same time to target cells, which enables it to kill target cells with a higher cytotoxicity than monomeric antibody toxin. Dimeric antibody toxin should also be more stable than scFv toxin.Streptococcal protein G is known to bind to IgG. It has a serum albumin binding domain and three IgG binding domains. It has been identified that the third IgG binding domain (domain III) of Streptococcal protein G binds to the Fab fragment of IgG (11).In this study, we focused on producing the dimer from the monomers that had already been refolded and purified. We constructed recombinant tandem repeats of Streptococcal protein G domain III and used it to make complexes that have multiple monomers bound. We found...

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“…This includes complement C1q, which activates the classical complement pathway (34), the neonatal FcR, which transports maternal IgG to the fetus (35), and FcgR, which triggers Ab-dependent cellular cytotoxicity, phagocytosis, secretion of inflammatory mediators, and Ag presentation (4,36). IgG Fc is also the target of several bacterial proteins including staphylococcal protein A (37) and streptococcal protein G (38,39). Of the four human IgG subclasses, the g-1 subclass is the most abundant in blood, displays the highest affinity to multiple FcgRs (4), and is most efficient at complement fixation and Ab-dependent cell-mediated killing (40,41).…”

Section: Discussionmentioning

confidence: 99%