Establishment of distinct MyoD, E2A, and twist DNA binding specificities by different basic region-DNA conformations

Basic helix-loop-helix (bHLH) proteins perform a wide variety of biological functions. Most bHLH proteins recognize the consensus DNA sequence CAN NTG (the E-box consensus sequence is underlined) but acquire further functional specificity by preferring distinct internal and flanking bases. In addition, induction of myogenesis by MyoD-related bHLH proteins depends on myogenic basic region (BR) and BR-HLH junction residues that are not essential for binding to a muscle-specific site, implying that their BRs may be involved in other critical interactions. We have investigated whether the myogenic residues influence DNA sequence recognition and how MyoD, Twist, and their E2A partner proteins prefer distinct CAN NTG sites. In MyoD, the myogenic BR residues establish specificity for particular CAN NTG sites indirectly, by influencing the conformation through which the BR helix binds DNA. An analysis of DNA binding by BR and junction mutants suggests that an appropriate BR-DNA conformation is necessary but not sufficient for myogenesis, supporting the model that additional interactions with this region are important. The sequence specificities of E2A and Twist proteins require the corresponding BR residues. In addition, mechanisms that position the BR allow E2A to prefer distinct half-sites as a heterodimer with MyoD or Twist, indicating that the E2A BR can be directed toward different targets by dimerization with different partners. Our findings indicate that E2A and its partner bHLH proteins bind to CAN NTG sites by adopting particular preferred BR-DNA conformations, from which they derive differences in sequence recognition that can be important for functional specificity.     

A protein target site in an early replicated human DNA sequence: a highly conserved binding motif

We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions.     

Crystal structure of PHO4 bHLH domain-DNA complex: flanking base recognition.

The crystal structure of a DNA-binding domain of PHO4 complexed with DNA at 2.8 A resolution revealed that the domain folds into a basic-helix-loop-helix (bHLH) motif with a long but compact loop that contains a short alpha-helical segment. This helical structure positions a tryptophan residue into an aromatic cluster so as to make the loop compact. PHO4 binds to DNA as a homodimer with direct reading of both the core E-box sequence CACGTG and its 3'-flanking bases. The 3'-flanking bases GG are recognized by Arg2 and His5. The residues involved in the E-box recognition are His5, Glu9 and Arg13, as already reported for bHLH/Zip proteins MAX and USF, and are different from those recognized by bHLH proteins MyoD and E47, although PHO4 is a bHLH protein.     

SREBP-1 dimerization specificity maps to both the helix-loop-helix and leucine zipper domains: use of a dominant negative

The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequence-specific DNA to regulate the expression of genes involved in lipid metabolism. We have designed a dominant negative (DN), termed A-SREBP-1, that inhibits the DNA binding of either SREBP protein. A-SREBP-1 consists of the dimerization domain of B-SREBP-1 and a polyglutamic acid sequence that replaces the basic region. A-SREBP-1 heterodimerizes with either B-SREBP-1 or B-SREBP-2, and both heterodimers are more stable than B-SREBP-1 bound to DNA. Circular dichroism thermal denaturation studies show that the B-SREBP-1.A-SREBP-1 heterodimer is -9.8 kcal mol(-1) dimer(-1) more stable than the B-SREBP-1 homodimer. EMSA assays demonstrate that A-SREBP-1 can inhibit the DNA binding of either B-SREBP-1 or B-SREBP-2 in an equimolar competition but does not inhibit the DNA binding of the three B-HLH-ZIP proteins MAX, USF, or MITF, even at 100 molar eq. Chimeric proteins containing the HLH domain of SREBP-1 and the leucine zipper from either MAX, USF, or MITF indicate that both the HLH and leucine zipper regions of SREBP-1 contribute to its dimerization specificity. Transient co-transfection studies demonstrate that A-SREBP-1 can inhibit the transactivation of SREBP-1 and SREBP-2 but not USF. A-SREBP-1 may be useful in metabolic diseases where SREBP family members are overexpressed.     

Classification of proteins based on the properties of the ligand-binding site: the case of adenine-binding proteins

Comparative analysis of protein binding sites for similar ligands yields information about conserved interactions, relevant for ligand affinity, and variable interactions, which are important for specificity. The pattern of variability can indicate new targets for a pharmacologically validated class of compounds binding to a similar site. A particularly vast group of therapeutically interesting proteins using the same or similar substrates are those that bind adenine-containing ligands. Drug development is focusing on compounds occupying the adenine-binding site and their specificity is an issue of paramount importance. We use a simple scheme to characterize and classify the adenine-binding sites in terms of their intermolecular interactions, and show that this classification does not necessarily correspond to protein classifications based on either sequence or structural similarity. We find that only a limited number of the different hydrogen bond patterns possible for adenine-binding is used, which can be utilized as an effective classification scheme. Closely related protein families usually share similar hydrogen patterns, whereas non-polar interactions are less well conserved. Our classification scheme can be used to select groups of proteins with a similar ligand-binding site, thus facilitating the definition of the properties that can be exploited to design specific inhibitors.