Interactions between a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus and a human κ light chain

The placement of a tryptophan residue into a single Ig-binding-domain of protein L from Peptostreptococcus magnus has been used to examine the binding interactions between the binding domain and ĸ light chains (ĸ-chains). The fluorescence intensity of the mutant domain increases on the formation of a complex with ĸ-chains. This has been used to determine the Kd of the complex under a range of conditions by using both pre-equilibrium and equilibrium methods. The Kd values determined for the complex with ĸ-chains at a number of different pH values are very close to those obtained with the wild-type domain, indicating that the mutation has not substantially affected its binding properties. Examination of the reaction between the mutant domain and ĸ-chains by stopped-flow fluorescence shows that complex formation takes place by two discrete, sequential processes. A fast bimolecular reaction, with a rate constant of 8.3×105 M-1·s-1 (at pH 8.0 and 25 °C), is followed by a slow unimolecular process with a rate (1.45 s-1) that is independent of the concentration of the reactants. This suggests that a conformational change occurs after the initial encounter complex is formed. The dissociation of the complex at equilibrium occurs in a single process of rate 0.095 s-1 at pH 8.0 and 25 °C. Stopped-flow CD studies show that a slow decrease in ellipticity at 275 nm occurs with a rate of 1.3 s-1 when wild-type protein binds to ĸ-chains, suggesting that the conformational transition might involve a change in environment around one or more tyrosine residues.

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Immunoglobulin-binding domains: Protein L from Peptostreptococcus magnus

Biochemical Society Transactions ◽

10.1042/bst0310716 ◽

2003 ◽

Vol 31 (3) ◽

pp. 716-718 ◽

Cited By ~ 22

Author(s):

N.G. Housden ◽

S. Harrison ◽

S.E. Roberts ◽

J.A. Beckingham ◽

M. Graille ◽

...

Keyword(s):

Binding Sites ◽

Protein A ◽

Cell Wall Protein ◽

Protein G ◽

Protein L ◽

Heavy Chains ◽

Binding Domains ◽

Peptostreptococcus Magnus ◽

Immunoglobulin Binding

Protein L is a multidomain cell-wall protein isolated from Peptostreptococcus magnus. It belongs to a group of proteins that contain repeated domains that are able to bind to Igs without stimulating an immune response, the most characterized of this group being Protein A (Staphylococcus aureus) and Protein G (Streptococcus). Both of these proteins bind predominantly to the interface of CH2-CH3 heavy chains, while Protein L binds exclusively to the VL domain of the κ-chain. The function of these proteins in vivo is not clear but it is thought that they enable the bacteria to evade the host's immune system. Two binding sites for κ-chain on a single Ig-binding domain from Protein L have recently been reported and we give evidence that one site has a 25–55-fold higher affinity for κ-chain than the second site.

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Mechanical stability analysis of the protein L immunoglobulin-binding domain by full alanine screening using molecular dynamics simulations

Biotechnology Journal ◽

10.1002/biot.201400231 ◽

2015 ◽

Vol 10 (3) ◽

pp. 386-394 ◽

Cited By ~ 3

Author(s):

Anna V. Glyakina ◽

Ilya V. Likhachev ◽

Nikolay K. Balabaev ◽

Oxana V. Galzitskaya

Keyword(s):

Molecular Dynamics ◽

Stability Analysis ◽

Molecular Dynamics Simulations ◽

Mechanical Stability ◽

Binding Domain ◽

Protein L ◽

Dynamics Simulations ◽

Immunoglobulin Binding

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Studies on a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus: the role of tyrosine-53 in the reaction with human IgG

Biochemical Journal ◽

10.1042/bj3530395 ◽

2001 ◽

Vol 353 (2) ◽

pp. 395-401 ◽

Cited By ~ 6

Author(s):

Jennifer A. BECKINGHAM ◽

Nicholas G. HOUSDEN ◽

Nicola M. MUIR ◽

Stephen P. BOTTOMLEY ◽

Michael G. GORE

Keyword(s):

Chain Complex ◽

Binding Domain ◽

Protein L ◽

Human Igg ◽

Tyrosine Residues ◽

Kd Values ◽

Trp Mutant ◽

The Stability ◽

Immunoglobulin Binding

Chemical modification experiments with tetranitromethane (TNM) have been used to investigate the role of tyrosine residues in the formation of the complex between PpL (the single Ig-binding domain of protein L, isolated from P. magnus strain 3316) and the kappa light chain (κ-chain). Reaction of PpL with TNM causes the modification of 1.9 equiv. of tyrosine (Tyr51 and Tyr53) and results in an approx. 140-fold decrease in affinity for human IgG. Similar experiments with mutated PpL proteins suggest that nitration predominantly inactivates the protein by modification of Tyr53. Reduction of the nitrotyrosine groups to aminotyrosine by incubation with sodium hydrosulphite does not restore high affinity for IgG. Modification of κ-chain by TNM resulted in the nitration of 3.1±0.09 tyrosine residues. When the PpLŐκ-chain complex was incubated with TNM, 4.1±0.04 tyrosine residues were nitrated, indicating that one tyrosine residue previously modified by the reagent was protected from TNM when the proteins are in complex with each other. The Kd for the equilibrium between PpL, human IgG and their complex has been shown by ELISA to be 112±20nM. A similar value (153±33nM) was obtained for the complex formed between IgG and the Tyr64 → Trp mutant (Y64W). However, the Kd values for the equilibria involving the PpL mutants Y53F and Y53F,Y64W were found to be 3.2±0.2 and 4.6±1µM respectively. These suggest that the phenol group of Tyr53 in PpL is important to the stability of the PpLŐκ-chain complex.

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