Microarray Profiling of Phage-Display Selections for Rapid Mapping of Transcription Factor–DNA Interactions

Modern computational methods are revealing putative transcription-factor (TF) binding sites at an extraordinary rate. However, the major challenge in studying transcriptional networks is to map these regulatory element predictions to the protein transcription factors that bind them. We have developed a microarray-based profiling of phage-display selection (MaPS) strategy that allows rapid and global survey of an organism's proteome for sequence-specific interactions with such putative DNA regulatory elements. Application to a variety of known yeast TF binding sites successfully identified the cognate TF from the background of a complex whole-proteome library. These factors contain DNA-binding domains from diverse families, including Myb, TEA, MADS box, and C2H2 zinc-finger. Using MaPS, we identified Dot6 as a trans-active partner of the long-predicted orphan yeast element Polymerase A & C (PAC). MaPS technology should enable rapid and proteome-scale study of bi-molecular interactions within transcriptional networks

Sequence-specific binding of normal serum immunoglobulin M to exposed protein C-termini

Both the timely clearance of degraded endogenous structures and the presence of secreted natural immunoglobulin M (IgM) are needed to avoid autoimmunity. These requirements may be causally related provided that natural IgM preferentially reacts with degraded antigens and, by activating complement, mediates their non-inflammatory clearance through complement receptors. We have previously shown that normal serum IgM reacts in vivo and in vitro with virtually all randomly generated C-terminal peptides displayed on T7 phage. The resultant multivalent IgM–peptide complexes activate complement and are detected by a loss of phage infectivity. A striking feature of these reactions is that different C-terminal peptides (≈ 3–4 amino acids) specifically react with different ‘C-terminal’ IgM (C-IgM) antibodies. This suggests that degraded supramolecular structures, expressing elevated levels of identical C-termini as a result of proteolysis, denaturation and abnormal exposure of repetitive protein constituents, may be preferential targets of C-IgM-mediated complement activation in the physiological environment. The specificity of C-IgM–peptide reactions is much higher than one would expect, assuming that normal serum IgM mostly comprises polyspecific natural antibodies. However, it is possible that polyspecific IgM is not adequately registered by our ‘functional’ phage-inactivation assays. In this study, we resolve the issue of C-IgM specificity by directly characterizing the binding reactivity of normal serum IgM with phage-displayed C-terminal peptides

Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches*

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca2+ and Mn2+. Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors