Structure of the leucine zipper.

In the basic-region leucine-zipper domain, flexible DNA-binding arms are juxtaposed by a two-stranded, parallel coiled-coil motif called the leucine zipper. Genetic, physical and structural studies of the leucine zipper identify interactions that help determine the stability and specificity of dimerization and DNA binding.     

Study of highly constitutively active mutants suggests how cAMP activates cAMP receptor protein

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.     

The role of helix stabilizing residues in GCN4 basic region folding and DNA binding

Basic region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a coiled-coil leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The basic region is largely unfolded in the absence of DNA, but adopts a helical conformation upon DNA binding. Although a coil --> helix transition is entropically unfavorable, this conformational change positions the DNA-binding residues appropriately for sequence-specific interactions with DNA. The N-terminal residues of the GCN4 DNA-binding domain, DPAAL, make no DNA contacts and are not part of the conserved basic region, but are nonetheless important for DNA binding. Asp and Pro are often found at the N-termini of alpha-helices, and such N-capping motifs can stabilize alpha-helical structure. In the present study, we investigate whether these two residues serve to stabilize a helical conformation in the GCN4 basic region, lowering the energetic cost for DNA binding. Our results suggest that the presence of these residues contributes significantly to helical structure and to the DNA-binding ability of the basic region in the absence of the leucine zipper. Similar helix-capping motifs are found in approximately half of all bZip domains, and the implications of these findings for in vivo protein function are discussed.     

Activity of the human centrosomal kinase, Nek2, depends on an unusual leucine zipper dimerization motif.

Nek2 is a human cell cycle-regulated kinase that is structurally related to the mitotic regulator, NIMA, of Aspergillus nidulans. Localization studies have shown that Nek2 is a core component of the centrosome, the microtubule organizing center of the cell, and functional approaches suggest a possible role for Nek2 in centrosome separation at the G2/M transition. Here, we have investigated the importance of an unusual leucine zipper coiled-coil motif present in the C-terminal noncatalytic domain of the Nek2 kinase. Glycerol gradient centrifugation indicated that endogenous Nek2 is present in HeLa cells as a salt-resistant 6 S complex, the predicted size of a Nek2 homodimer. Recombinant Nek2 overexpressed in insect cells also formed a 6 S complex, whereas a Nek2 mutant specifically lacking the leucine zipper motif was monomeric. Using yeast two-hybrid interaction analyses and coprecipitation assays, we found that Nek2 can indeed form homodimers both in vivo and in vitro and that this dimerization specifically required the leucine zipper motif. Moreover, deletion of the leucine zipper prevented a trans-autophosphorylation reaction on the C-terminal domain of Nek2 and strongly reduced Nek2 kinase activity on exogenous substrates. Finally, we emphasize that the Nek2 leucine zipper described here differs from classical leucine zippers in that it exhibits a radically different arrangement of hydrophobic and charged amino acids. Thus, this study reveals not only an important mechanism for the regulation of the Nek2 kinase but, furthermore, highlights an unusual organization of a leucine zipper dimerization motif.     

A C-helix residue, Arg-123, has important roles in both the active and inactive forms of the cAMP receptor protein

The cAMP receptor protein (CRP) of Escherichia coli exists in an equilibrium between active and inactive forms, and the effector, cAMP, shifts that equilibrium to the active form, thereby allowing DNA binding. For this equilibrium shift, a C-helix repositioning around the C-helix residues Thr-127 and Ser-128 has been reported as a critical local event along with proper beta4/beta5 positioning. Here we show that another C-helix residue, Arg-123, has a unique role in cAMP-dependent CRP activation in two different ways. First, Arg-123 is important for proper cAMP affinity, although it is not critical for the conformational change with saturating amounts of cAMP. Second, Arg-123 is optimal for stabilizing the inactive conformation of CRP when cAMP is absent, thereby allowing a maximal range of regulation by cAMP. However, Arg-123 does not appear to be critical for a functional response to cAMP, as has been proposed previously (Berman, H. M., Ten Eyck, L. F., Goodsell, D. S., Haste, N. M., Korney, A., and Taylor, S. S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 45-50). Based on mutagenic evidence, we also propose the basis for the stabilization of the inactive form to be through a salt interaction between Asp-68 and Arg-123.     

Mechanism of the CO-sensing heme protein CooA: new insights from the truncated heme domain and UVRR spectroscopy

The bacterial CO-sensing heme protein CooA activates expression of genes whose products perform CO-metabolism by binding its target DNA in response to CO binding. The required conformational change has been proposed to result from CO-induced displacement of the heme and of the adjacent C-helix, which connects the sensory and DNA-binding domains. Support for this proposal comes from UV Resonance Raman (UVRR) spectroscopy, which reveals a more hydrophobic environment for the C-helix residue Trp110 when CO binds. In addition, we find a tyrosine UVRR response, which is attributable to weakening of a Tyr55-Glu83 H-bond that anchors the proximal side of the heme. Both Trp and Tyr responses are augmented in the heme domain when the DNA-binding domain has been removed, apparently reflecting loss of the inter-domain restraint. This augmentation is abolished by a Glu83Gln substitution, which weakens the anchoring H-bond. The CO recombination rate following photolysis of the CO adduct is similar for truncated and full-length protein, though truncation does increase the rate of CO association in the absence of photolysis; together these data indicate that truncation causes a faster dissociation of the endogenous Pro2 ligand. These findings are discussed in the light of structural evidence that the N-terminal tail, once released from the heme, selects the proper orientation of the DNA-binding domain, via docking interactions.