Enzymatic Cleavage of a CD4 Immunoadhesin Generates Crystallizable, Biologically Active Fd-like Fragments

Abstract

CD4, the cell-surface receptor for the human immunodeficiency virus (HIV), is a member of the immunoglobulin (Ig) gene superfamily. It contains four extracellular sequences homologous to Ig V_L, domains. The first of these (V_1) is sufficient for binding to HIV; however, the structural basis for this binding has yet to be elucidated. While several models for the structure of Ig-like domains in CD4 have been proposed on the basis of crystal structures of Ig V_1, domains, direct evidence that CD4 and VL domains fold similarly has not been obtained. To produce individual domains of CD4 for structural studies, we used molecular fusions of such domains with Ig heavy chain (CD4 immunoadhesins), which are very efficiently expressed and secreted in mammalian cells and can be easily isolated in single-step purification with protein A. Since these fusion molecules are antibody-like homodimeric proteins, we investigated the possibility that they might be cleaved enzymatically to produce Fd-like and Fc fragments. We found that cleavage with papain releases an Fd-like fragment containing the V_1 and V_2 CD4 domains: this fragment fully retains the ability to bind to the HIV-1 envelope glycoprotein gp120 and to block HIV infection in vitro. Moreover, folding of the CD4 domains in the Fd-like fragment and in the parent immunoadhesin is indistinguishable, as indicated by circular dichroism. Spectral analysis of the Fd-like fragment suggests that secondary structure content is identical with that predicted from the known structure of Ig V_L domains; this directly supports the hypothesis that the V_1 and V_2 domains of CD4 fold similarly to Ig V_L domains. Crystals of the Fd-like fragment diffract beyond 3-Å resolution and are suitable for detailed structural analysis. The approach described here may be useful, as an alternative to direct expression, in the study of receptors and other adhesion molecules which are members of the Ig gene superfamily.

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