Cysteine 50 of the POU H domain determines the range of targets recognized by POU proteins

Abstract

The best target of POU proteins (Oct-1, Oct-2) is an octamer sequence ATGCAAAT. POU proteins also recognize, with weaker affinity, the TAAT-like targets of another group of regulatory factors, the homeoproteins. Up to now, it has not been known why Cys50 of the POUHdomain is absolutely conserved in contrast to that in homeoproteins. To assess the importance of Cys50 in determining the binding specificity of POU proteins, all possible amino acids were substituted for Cys at position 50, and the resulting mutants were tested with probes containing octamer (ATGCAAATNN) or homeospecific binding sites. Only the wild-type POU was shown to adequately discriminate between the octamer and homeospecific sites, and the protein affinity was only slightly affected by the nucleotide sequence flanking the octamer at the 3'-end. Any amino acid substitution at position 50 resulted in the mutant protein binding efficiently both to the octamer and the TAAT-like sequences. Moreover, in this case the 3'-flanking sequences influenced the binding to a much greater extent.

The role of the annexin A2 heterotetramer in vascular fibrinolysis

“…Presently, a Cys-binding motif has not been identified in any protein, although cysteine residues of proteins interact noncovalently with certain nucleotide sequences. 96 The discrepancy between these results and ours could be explained by the structure of the recombinant ANXA2 used in these studies. The ANXA2, which did not bind tPA, was a recombinant protein (Ser-1-Asp-338) whose structure and function were shown to be identical to tissue ANXA2, 43 whereas the recombinant protein that bound tPA was a fusion protein consisting of an N-terminal tag (MASMTGGQQMGRDP) and a C-terminal tag (LEHHHHHH).…”

Isoforms of Hepatocyte Nuclear Factor-6 Differ in DNA-binding Properties, Contain a Bifunctional Homeodomain, and Define the New ONECUT Class of Homeodomain Proteins

“…20). Mutation of this amino acid can modify the sequence specificity as in, for example, the case in Bicoid (19) or alter the discrimination among probes, for example, as in POU factors (45) or else leave the DNA-binding properties unaffected as the Q50A mutation in the engrailed homeodomain (18). We found here that the homeodomain of HNF-6 is required for binding to probes such as the TTR probe.…”

Tissue-specific isoforms of the ubiquitous transcription factor Oct-1

“…Cooperative interaction with Oct-1 has been shown for several transcription factors: Sp1 (Janson and Petterson 1990;Strom et al 1996), Ap-1 (Kaushansky et al 1994;Delhase et al 1996), NF-1 (O'Connor and Bernard 1995), steroid hormone receptor (Bruggemeier et al 1991), Pit-1 (Delhase et al 1996), and the homeobox protein Pbx (Subramaniam et al 1998). Second, pleiotropic functioning of Oct-1 and ®ne regulation of expression may proceed by interaction of Oct-1 with various DNA sites: the canonical oct site ATGCAAAT, non-canonical oct sites with single nucleotide substitutions, TAAT core sites, and TAATGARAT sites (Verrijzer et al 1992;Stepchenko 1992bStepchenko , 1994Stepchenko et al 1997aStepchenko et al , 1997b. The anity of Oct-1 for these sites is highly variable, and interaction with the co-activator OCA-B (Gstaiger et al 1995;Luo and Roeder 1995;Strubin et al 1995) and with VP-16 (O'Hare and Goding 1988) strictly depends on the site to which Oct-1 is bound.…”

“…We previously found A/T-rich clusters with TAAT core sites in this 5¢-terminal region. These sites are known to bind Oct-1 and Oct-2 proteins (Verrijzer et al 1992;Stepchenko 1992bStepchenko , 1994Stepchenko et al 1997aStepchenko et al , 1997bPankratova and Polanovsky 1998). Most of these TAAT core sites were also found in human DNA.…”