Structural studies in solution of the recombinant N-terminal pair of short consensus/complement repeat domains of complement receptor type 2 (CR2/CD21) and interactions with its ligand C3dg

Joel M. Guthridge, Jonathan K. Rakstang, Kendra A. Young, Justin Hinshelwood, Mohammed Aslam, Alexis Robertson, Matthew G. Gipson, Maria Rossa Sarrias, William T. Moore, Michael Meagher, David Karp, John D. Lambris, Stephen J. Perkins, V. Michael Holers

Research output: Contribution to journalArticle

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Abstract

Human complement receptor type 2 (CR2, CD21) is a cell surface receptor that binds three distinct ligands (complement C3d, Epstein-Barr virus gp350/220, and the low-affinity IgE receptor CD23) via the N-terminal two of fifteen or sixteen short consensus/complement repeat (SCR) domains. Here, we report biophysical studies of the CR2 SCR 1-2 domain binding to its ligand C3dg. Two recombinant forms of CR2 containing the SCR 1-2 and SCR 1-15 domains were expressed in high yield in Pichia pastoris and baculovirus, respectively. Circular dichroism spectroscopy showed that CR2 SCR 1-2 receptor possessed a β-sheet secondary structure with a melting temperature of 59 °C. Using surface plasmon resonance, kinetic parameters for the binding of either CR2 SCR 1-2 or the full-length SCR 1-15 form of CR2 showed that the affinity of binding to immobilized C3d is comparable for the SCR 1-15 compared to the SCR 1-2 form of CR2. Unexpectedly, both the association and dissociation rates for the SCR 1-15 form were slower than for the SCR 1-2 form. These data show that the SCR 1-2 domains account for the primary C3dg binding site of CR2 and that the additional SCR domains of full-length CR2 influence the ability of CR2 SCR 1-2 to interact with its ligand. Studies of the pH and ionic strength dependence of the interaction between SCR 1-2 and C3d by surface plasmon resonance showed that this is influenced by charged interactions, possibly involving the sole His residue in CR2 SCR 1-2. Sedimentation equilibrium studies of CR2 SCR 1-2 gave molecular weights of 17 000, in good agreement with its sequence-derived molecular weight to show that this was monomeric. Its sedimentation coefficient was determined to be 1.36 S. The complex with C3d gave molecular weights in 50 mM and 200 mM NaCl buffer that agreed closely with its sequence-derived molecular weight of 50 600 and showed that a 1:1 complex had been formed. Molecular graphics views of homology models for the separate CR2 SCR 1 and SCR 2 domains showed that both SCR domains exhibited a distribution of charged groups throughout its surface. The single His residue is located near a long eight-residue linker between the two SCR domains and may influence the linker conformation and the association of C3d and CR2 SCR 1-2 into their complex. Sedimentation modeling showed that the arrangement of the two SCR domains in CR2 SCR 1-2 is highly extended in solution.

Original languageEnglish (US)
Pages (from-to)5931-5941
Number of pages11
JournalBiochemistry
Volume40
Issue number20
DOIs
StatePublished - May 22 2001

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Complement 3d Receptors
Complement C1
Molecular weight
Sedimentation
Ligands
Surface plasmon resonance
Molecular graphics
Complement C3d
Association reactions
Circular dichroism spectroscopy
IgE Receptors
Cell Surface Receptors
Ionic strength
Viruses
Kinetic parameters
Melting point
Conformations
Buffers
Binding Sites
Molecular Weight

ASJC Scopus subject areas

  • Biochemistry

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Structural studies in solution of the recombinant N-terminal pair of short consensus/complement repeat domains of complement receptor type 2 (CR2/CD21) and interactions with its ligand C3dg. / Guthridge, Joel M.; Rakstang, Jonathan K.; Young, Kendra A.; Hinshelwood, Justin; Aslam, Mohammed; Robertson, Alexis; Gipson, Matthew G.; Sarrias, Maria Rossa; Moore, William T.; Meagher, Michael; Karp, David; Lambris, John D.; Perkins, Stephen J.; Holers, V. Michael.

In: Biochemistry, Vol. 40, No. 20, 22.05.2001, p. 5931-5941.

Research output: Contribution to journalArticle

Guthridge, JM, Rakstang, JK, Young, KA, Hinshelwood, J, Aslam, M, Robertson, A, Gipson, MG, Sarrias, MR, Moore, WT, Meagher, M, Karp, D, Lambris, JD, Perkins, SJ & Holers, VM 2001, 'Structural studies in solution of the recombinant N-terminal pair of short consensus/complement repeat domains of complement receptor type 2 (CR2/CD21) and interactions with its ligand C3dg', Biochemistry, vol. 40, no. 20, pp. 5931-5941. https://doi.org/10.1021/bi0101749
Guthridge, Joel M. ; Rakstang, Jonathan K. ; Young, Kendra A. ; Hinshelwood, Justin ; Aslam, Mohammed ; Robertson, Alexis ; Gipson, Matthew G. ; Sarrias, Maria Rossa ; Moore, William T. ; Meagher, Michael ; Karp, David ; Lambris, John D. ; Perkins, Stephen J. ; Holers, V. Michael. / Structural studies in solution of the recombinant N-terminal pair of short consensus/complement repeat domains of complement receptor type 2 (CR2/CD21) and interactions with its ligand C3dg. In: Biochemistry. 2001 ; Vol. 40, No. 20. pp. 5931-5941.
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T1 - Structural studies in solution of the recombinant N-terminal pair of short consensus/complement repeat domains of complement receptor type 2 (CR2/CD21) and interactions with its ligand C3dg

AU - Guthridge, Joel M.

AU - Rakstang, Jonathan K.

AU - Young, Kendra A.

AU - Hinshelwood, Justin

AU - Aslam, Mohammed

AU - Robertson, Alexis

AU - Gipson, Matthew G.

AU - Sarrias, Maria Rossa

AU - Moore, William T.

AU - Meagher, Michael

AU - Karp, David

AU - Lambris, John D.

AU - Perkins, Stephen J.

AU - Holers, V. Michael

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N2 - Human complement receptor type 2 (CR2, CD21) is a cell surface receptor that binds three distinct ligands (complement C3d, Epstein-Barr virus gp350/220, and the low-affinity IgE receptor CD23) via the N-terminal two of fifteen or sixteen short consensus/complement repeat (SCR) domains. Here, we report biophysical studies of the CR2 SCR 1-2 domain binding to its ligand C3dg. Two recombinant forms of CR2 containing the SCR 1-2 and SCR 1-15 domains were expressed in high yield in Pichia pastoris and baculovirus, respectively. Circular dichroism spectroscopy showed that CR2 SCR 1-2 receptor possessed a β-sheet secondary structure with a melting temperature of 59 °C. Using surface plasmon resonance, kinetic parameters for the binding of either CR2 SCR 1-2 or the full-length SCR 1-15 form of CR2 showed that the affinity of binding to immobilized C3d is comparable for the SCR 1-15 compared to the SCR 1-2 form of CR2. Unexpectedly, both the association and dissociation rates for the SCR 1-15 form were slower than for the SCR 1-2 form. These data show that the SCR 1-2 domains account for the primary C3dg binding site of CR2 and that the additional SCR domains of full-length CR2 influence the ability of CR2 SCR 1-2 to interact with its ligand. Studies of the pH and ionic strength dependence of the interaction between SCR 1-2 and C3d by surface plasmon resonance showed that this is influenced by charged interactions, possibly involving the sole His residue in CR2 SCR 1-2. Sedimentation equilibrium studies of CR2 SCR 1-2 gave molecular weights of 17 000, in good agreement with its sequence-derived molecular weight to show that this was monomeric. Its sedimentation coefficient was determined to be 1.36 S. The complex with C3d gave molecular weights in 50 mM and 200 mM NaCl buffer that agreed closely with its sequence-derived molecular weight of 50 600 and showed that a 1:1 complex had been formed. Molecular graphics views of homology models for the separate CR2 SCR 1 and SCR 2 domains showed that both SCR domains exhibited a distribution of charged groups throughout its surface. The single His residue is located near a long eight-residue linker between the two SCR domains and may influence the linker conformation and the association of C3d and CR2 SCR 1-2 into their complex. Sedimentation modeling showed that the arrangement of the two SCR domains in CR2 SCR 1-2 is highly extended in solution.

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