Ion Binding Properties of a Naturally Occurring Metalloantibody
Elinaz Farokhi,
Jonathan K. Fleming,
M. Frank Erasmus,
Aaron D. Ward,
Yunjin Wu,
Maria G. Gutierrez,
Jonathan M. Wojciak,
Tom Huxford
Affiliations
Elinaz Farokhi
Structural Biochemistry Laboratory, Department of Chemistry & Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-1030, USA
Jonathan K. Fleming
Structural Biochemistry Laboratory, Department of Chemistry & Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-1030, USA
M. Frank Erasmus
Structural Biochemistry Laboratory, Department of Chemistry & Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-1030, USA
Aaron D. Ward
Structural Biochemistry Laboratory, Department of Chemistry & Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-1030, USA
Yunjin Wu
Structural Biochemistry Laboratory, Department of Chemistry & Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-1030, USA
Maria G. Gutierrez
Structural Biochemistry Laboratory, Department of Chemistry & Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-1030, USA
Jonathan M. Wojciak
Apollo Endosurgery, Inc. (formerly Lpath, Inc.) 1120 S. Capital of Tx Hwy, Bldg. 1, Suite 300, Austin, TX 78746, USA
Tom Huxford
Structural Biochemistry Laboratory, Department of Chemistry & Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-1030, USA
LT1009 is a humanized version of murine LT1002 IgG1 that employs two bridging Ca2+ ions to bind its antigen, the biologically active lipid sphingosine-1-phosphate (S1P). We crystallized and determined the X-ray crystal structure of the LT1009 Fab fragment in 10 mM CaCl2 and found that it binds two Ca2+ in a manner similar to its antigen-bound state. Flame atomic absorption spectroscopy (FAAS) confirmed that murine LT1002 also binds Ca2+ in solution and inductively-coupled plasma-mass spectrometry (ICP-MS) revealed that, although Ca2+ is preferred, LT1002 can bind Mg2+ and, to much lesser extent, Ba2+. Isothermal titration calorimetry (ITC) indicated that LT1002 binds two Ca2+ ions endothermically with a measured dissociation constant (KD) of 171 μM. Protein and genome sequence analyses suggested that LT1002 is representative of a small class of confirmed and potential metalloantibodies and that Ca2+ binding is likely encoded for in germline variable chain genes. To test this hypothesis, we engineered, expressed, and purified a Fab fragment consisting of naïve murine germline-encoded light and heavy chain genes from which LT1002 is derived and observed that it binds Ca2+ in solution. We propose that LT1002 is representative of a class of naturally occurring metalloantibodies that are evolutionarily conserved across diverse mammalian genomes.
