Abstract
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.
Highlights
At least one-third, and perhaps as many as one-half, of all functioning proteins are predicted to be metalloproteins [1,2,3]
Our results suggest that the anti-S1P antibody does not require antigen in order to bind Ca2+ and that the default Ca2+ binding mode is similar to that observed in the LT1009:Ca2+ :S1P complex X-ray crystal structure
By crystallizing and determining the X-ray crystal structure of the Q425 Fab fragment separately in the presence of 10 mM Ba2+, 10 mM Ca2+, and 10 mM EDTA, researchers concluded that one Ca2+ binds to a site at the interface between the light and heavy chains, employing amino acid side
Summary
At least one-third, and perhaps as many as one-half, of all functioning proteins are predicted to be metalloproteins [1,2,3]. Included among these metal-dependent biological factors are the vast collection of diverse metalloenzymes, which include many oxidoreductases, proteases, and all protein kinases, as well as metal-dependent extracellular receptors, signaling proteins, transcription factors, and charge transport complexes [4]. In 2009, as part of an effort aimed at developing novel anti-inflammatory and potential anticancer antibody-based therapies that function by selectively binding to signaling lipids, we determined the X-ray crystal structure of the Fab fragment of a humanized mouse monoclonal antibody bound to its antigen sphingosine-1-phosphate (S1P) [5,6]. The two Ca2+ are partially coordinated within close proximity (
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