Steady state kinetics and inhibition by a dipyridodiazepinone of the reverse transcriptase from human immunodeficiency virus type 1 (HIV) were studied using a heteropolymeric RNA template with a sequence from the authentic initiation site on the HIV genome. For addition of the first deoxynucleotide to primer, kcat/KM is 0.05 (nM-min)-1 and KM is 10 nM. When all 4 deoxynucleotide triphosphates are present and processive synthesis occurs, catalysis is less efficient; kcat/KM = .0077 (nM-min)-1 and KM = 100 nM for dATP. These results are consistent with a rate determining conformation change involved in translocation of the enzyme along the template. Inhibition by the dipyridodiazepinone BI-RG-587 is noncompetitive with respect to both nucleotide and template-primer; this compound decreases Vmax but does not affect KM. Thus, this inhibitor binds to a site distinct from the substrate binding sites with Ki of 220 nM. Inhibition by BI-RG-587 results in a uniform decrease in amount of products of all lengths rather than a shift from longer to shorter products, suggesting the inhibitor does not affect processivity of reverse transcriptase.
Myeloid-derived growth factor (MYDGF) is a paracrine-acting protein that is produced by bone marrow-derived monocytes and macrophages to protect and repair the heart after myocardial infarction (MI). This effect can be used for the development of protein-based therapies for ischemic tissue repair, also beyond the sole application in heart tissue. Here, we report the X-ray structure of MYDGF and identify its functionally relevant receptor binding epitope. MYDGF consists of a 10-stranded β-sandwich with a folding topology showing no similarities to other cytokines or growth factors. By characterizing the epitope of a neutralizing antibody and utilizing functional assays to study the activity of surface patch-mutations, we were able to localize the receptor interaction interface to a region around two surface tyrosine residues 71 and 73 and an adjacent prominent loop structure of residues 97–101. These findings enable structure-guided protein engineering to develop modified MYDGF variants with potentially improved properties for clinical use.
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