Interfacial metal and antibody recognition

doi:10.1073/pnas.0507267102

Interfacial metal and antibody recognition

^*Vaccine Research Center, National Institute of Allergy and Infectious Diseases, and ^†National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; ^‡Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, New York, NY 10032; and ^¶The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom

Contributed by Wayne A. Hendrickson, August 22, 2005

Abstract

The unique ligation properties of metal ions are widely exploited by proteins, with approximately one-third of all proteins estimated to be metalloproteins. Although antibodies use various mechanisms for recognition, to our knowledge, none has ever been characterized that uses an interfacial metal. We previously described a family of CD4-reactive antibodies, the archetype being Q425. CD4:Q425 engagement does not interfere with CD4:HIV-1 gp120 envelope glycoprotein binding, but it blocks subsequent steps required for viral entry. Here, we use surface-plasmon resonance to show that Q425 requires calcium for recognition of CD4. Specifically, Q425 binding of calcium resulted in a 55,000-fold enhancement in affinity for CD4. X-ray crystallographic analyses of Q425 in the presence of Ca²⁺, Ba²⁺, or EDTA revealed an exposed metal-binding site, partially coordinated by five atoms contributed from four antibody complementarity-determining regions. The results suggest that Q425 recognition of CD4 involves direct ligation of antigen by the Q425-held calcium, with calcium binding each ligating atom of CD4 with ≈1.5 kcal/mol of binding energy. This energetic contribution, which is greater than that from a typical protein atom, demonstrates how interfacial metal ligation can play a unique role in antigen recognition.

Footnotes

↵ ^§To whom correspondence may be addressed. E-mail: wayne{at}convex.hhmi.columbia.edu. ^∥To whom correspondence may be addressed at: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Building 40, Room 4508, Bethesda, MD 20892-3027. E-mail: pdkwong{at}nih.gov.
Abbreviations: CDR, complementarity-determining region; Fab, antigen-binding Ab fragment; Fv, variable domain; PDB, Protein Data Bank; RU, response units; SPR, surface-plasmon resonance.
Data deposition: The atomic coordinates of Q425 in the presence of calcium, barium, and EDTA have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2ADJ, 2ADI, and 2ADG, respectively). The nucleotide sequences for Q425, Q428, and Q4116 have been deposited in the GenBank database (accession nos. DQ132636-DQ132641, respectively).
Freely available online through the PNAS open access option.