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IHF is a protein of the bacterial nucleoid proteins (NAPs for nucleoid-associated proteins) involved in DNA structuring and transcription regulation. In vivo interplay between different NAPs determines selectively the expression rate of many genes. Here, we show that IHF is a trans-acting factor implicated directly in the regulation of the proU promoter of Escherichia coli by binding specifically and solely around the promoter box. proU expression is mainly under the repression effect of another NAP, H-NS. We show that IHF binding to proU organize the promoter DNA local structure in a completely different way than H-NS binding. Thus, we propose that the partial alleviation of H-NS repression is mediated by the promoter structure modification.

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Mutations in the human erythroid Krüppel-like factor (EKLF) can lead to either anemia or the benign InLu phenotype. To elucidate the relationship between these mutations and the differing phenotypes, we prepared recombinant forms of wild-type and 5 mutant EKLF proteins and quantitated their binding affinity to a range of EKLF-regulated genes. Missense mutants (R328H, R328L, and R331G) from persons with InLu phenotype did not bind DNA. Hence, as with the heterozygous loss of function nonsense (L127X, S270X, and K292X) and frameshift (P190Lfs and R319Efs) EKLF mutations, monoallelic loss of EKLF does not result in haploinsufficiency at all loci. In contrast, K332Q has a slightly reduced DNA binding affinity (∼ 2-fold) for all promoters examined but exhibits a phenotype only in a compound heterozygote with a nonfunctional allele. E325K also has a reduced, but significant, binding affinity, particularly for the ß-globin gene but results in a disease phenotype even with the wild-type allele expressed, although not as a classic dominant-negative mutant. E325K protein may therefore actively interfere with EKLF-dependent processes by destabilizing transcription complexes, providing a rational explanation for the severity of the disease phenotype. Our study highlights the critical role of residues within the second EKLF zinc finger domain.

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Although DNA flexibility is known to play an important role in DNA-protein interactions, the importance of protein flexibility is less well understood. Here, we show that protein dynamics are important in DNA recognition using the well-characterized human papillomavirus (HPV) type 6 E2 protein as a model system. We have compared the DNA binding properties of the HPV 6 E2 DNA binding domain (DBD) and a mutant lacking two C-terminal leucine residues that form part of the hydrophobic core of the protein. Deletion of these residues results in increased specific and non-specific DNA binding and an overall decrease in DNA binding specificity. Using (15)N NMR relaxation and hydrogen/deuterium exchange, we demonstrate that the mutation results in increased flexibility within the hydrophobic core and loop regions that orient the DNA binding helices. Stopped-flow kinetic studies indicate that increased flexibility alters DNA binding by increasing initial interactions with DNA but has little or no effect on the structural rearrangements that follow this step. Taken together these data demonstrate that subtle changes in protein dynamics have a major influence on protein-DNA interactions.

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The E2 proteins are transcription/replication factors from papillomaviruses. Human papillomaviruses (HPVs) can be broadly divided in two groups; low-risk HPV subtypes cause benign warts while high-risk HPVs give rise to cervical cancer. Although a range of crystal structures of E2 DNA-binding domains (DBD) from both high- and low-risk HPV subtypes have been reported previously, structures of E2 DBD:DNA complexes have only been available for high-risk HPV18 and bovine papillomavirus (BPV1). In the present study we report the unliganded and DNA complex structures of the E2 DBD from the low-risk HPV6. As in the previous E2-DNA structures, complex formation results in considerable bending of the DNA, which is facilitated by sequences with A:T-rich spacers that adopt a pre-bent conformation. The low-risk HPV6 E2-DNA complex differs from the earlier structures in that minimal deformation of the protein accompanies complex formation. Stopped-flow kinetic studies confirm that both high- and low-risk E2 proteins adapt their structures on binding to DNA, although this is achieved more readily for HPV6 E2. It therefore appears that the higher selectivity of the HPV6 E2 protein may arise from its limited molecular adaptability, a property that might distinguish the behaviour of E2 proteins from high- and low-risk HPV subtypes.

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Malaria caused by Plasmodium vivax is a major cause of global morbidity and, in rare cases, mortality. Lactate dehydrogenase is an essential Plasmodium protein and, therefore, a potential antimalarial drug target. Ideally, drugs directed against this target would be effective against both major species of Plasmodium, P. falciparum and P. vivax. In this study, the crystal structure of the lactate dehydrogenase protein from P. vivax has been solved and is compared to the equivalent structure from the P. falciparum enzyme. The active sites and cofactor binding pockets of both enzymes are found to be highly similar and differentiate these enzymes from their human counterparts. These structures suggest effective inhibition of both enzymes should be readily achievable with a common inhibitor. The crystal structures of both enzymes have also been solved in complex with the synthetic cofactor APADH. The unusual cofactor binding site in these Plasmodium enzymes is found to readily accommodate both NADH and APADH, explaining why the Plasmodium enzymes retain enzymatic activity in the presence of this synthetic cofactor.

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