Stereochemical basis of DNA recognition by Zn fingers.

DNA-recognition rules for Zn fingers are discussed in terms of crystal structures. The rules can explain the DNA-binding characteristics of a number of Zn finger proteins for which there are no crystal structures. The rules have two parts: chemical rules, which list the possible pairings between the 4 DNA bases and the 20 amino acid residues, and stereochemical rules, which describe the specific base positions contacted by several amino acid positions in the Zn finger. It is discussed that to maintain the correct binding geometry, in which the N-terminus of the recognition helix is closer to the DNA than the C-terminus, the residues facing the DNA on the helix must be larger near the C-terminus, and that two different types of fingers (A and B) bind to DNA in distinctly different ways and cover different numbers of base pairs.     

A role for the passage helix in the DNA cleavage reaction of eukaryotic topoisomerase II. A two-site model for enzyme-mediated DNA cleavage.

Eukaryotic topoisomerase II is capable of binding two separate nucleic acid helices prior to its DNA cleavage and strand passage events (Zechiedrich, E. L., and Osheroff, N (1990) EMBO J. 9, 4555-4562). Presumably, one of these helices represents the helix that the enzyme cleaves (i.e. cleavage helix), and the other represents the helix that it passes (i.e. passage helix) through the break in the nucleic acid backbone. To determine whether the passage helix is required for reaction steps that precede the enzyme's DNA strand passage event, interactions between Drosophila melanogaster topoisomerase II and a short double-stranded oligonucleotide were assessed. These studies employed a 40-mer that contained a specific recognition/cleavage site for the enzyme. The sigmoidal DNA concentration dependence that was observed for cleavage of the 40-mer indicated that topoisomerase II had to interact with more than a single oligonucleotide in order for cleavage to take place. Despite this requirement, results of enzyme DNA binding experiments indicated no binding cooperativity for the 40-mer. These findings strongly suggest a two-site model for topoisomerase II action in which the passage and the cleavage helices bind to the enzyme independently, but the passage helix must be present for efficient topoisomerase II-mediated DNA cleavage to occur.     

Interaction of transmembrane helices by a knobs-into-holes packing characteristic of soluble coiled coils

Membrane-embedded protein domains frequently exist as α-helical bundles, as exemplified by photosynthetic reaction centers, bacteriorhodopsin, and cytochrome C oxidase. The sidechain packing between their transmembrane helices was investigated by a nearest-neighbor analysis which identified sets of interfacial residues for each analyzed helix–helix interface. For the left-handed helix–helix pairs, the interfacial residues almost exclusively occupy positions a, d, e, or g within a heptad motif (abcdefg) which is repeated two to three times for each interacting helical surface. The connectivity between the interfacial residues of adjacent helices conforms to the knobs-into-holes type of sidechain packing known from soluble coiled coils. These results demonstrate on a quantitative basis that the geometry of sidechain packing is similar for left-handed helix–helix pairs embedded in membranes and coiled coils of soluble proteins. The transmembrane helix–helix interfaces studied are somewhat less compact and regular as compared to soluble coiled coils and tolerate all hydrophobic amino acid types to similar degrees. The results are discussed with respect to previous experimental findings which demonstrate that specific interactions between transmembrane helices are important for membrane protein folding and/or oligomerization. Proteins 31:150–159, 1998. © 1998 Wiley-Liss, Inc.     

Genetic analysis of the LexA repressor: Isolation and characterization of LexA(Def) mutant proteins

Summary We report the isolation of LexA mutant proteins with impaired repressor function. These mutant proteins were obtained by transforming a LexA-deficient recA-lacZ indicator strain with a randomly mutagenized plasmid harbouring the lexA gene and subsequent selection on MacConkey-lactose indicator plates. A total of 24 different lexA(Def) missense mutations were identified. All except three mutant proteins are produced in near-normal amounts suggesting that they are fairly resistant to intracellular proteases. All lexA(Def) missense mutations are situated within the first 67 amino acids of the amino-terminal DNA binding domain. The properties of an intragenic deletion mutant suggest that the part of the amino-terminal domain important for DNA recognition or domain folding should extent at least to amino acids 69 or 70. A recent 2D-NMR study (Lamerichs et al. 1989) has identified three a helices in the DNA binding domain of LexA. The relative orientation of two of them (helices 2 and 3) is reminiscent of, but not identical to, the canonical helix-turn-helix motif suggesting nevertheless that helix 3 might be involved in DNA recognition. The distribution of the lexA(Def) missense mutations along the first 67 amino-terminal amino acids indeed shows some clustering within helix 3, since 8 out of the 24 different missense mutations are found in this helix. However one mutation in front of helix 1 and five mutations between amino acids 61 and 67 suggest that elements other than helices 2 and 3 may be important for DNA binding.     

High-resolution prediction of protein helix positions and orientations

We have developed a new method for predicting helix positions in globular proteins that is intended primarily for comparative modeling and other applications where high precision is required. Unlike helix packing algorithms designed for ab initio folding, we assume that knowledge is available about the qualitative placement of all helices. However, even among homologous proteins, the corresponding helices can demonstrate substantial differences in positions and orientations, and for this reason, improperly positioned helices can contribute significantly to the overall backbone root-mean-square deviation (RMSD) of comparative models. A helix packing algorithm for use in comparative modeling must obtain high precision to be useful, and for this reason we utilize an all-atom protein force field (OPLS) and a Generalized Born continuum solvent model. To reduce the computational expense associated with using a detailed, physics-based energy function, we have developed new hierarchical and multiscale algorithms for sampling the helices and flanking loops. We validate the method using a test suite of 33 cases, which are drawn from a diverse set of high-resolution crystal structures. The helix positions are reproduced with an average backbone RMSD of 0.6 A, while the average backbone RMSD of the complete loop-helix-loop region (i.e., the helix with the surrounding loops, which are also repredicted) is 1.3 A.     

Conformational analysis by NMR and distance geometry techniques of a peptide mimetic of the third helix of the Antennapedia homeodomain.

The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well-known helix-turn-helix motif. The recognition helix penetrates the DNA major groove, gives specific protein-DNA contacts and forms direct, or water-mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42-53, where residues W48 and F49, not involved in the protein-DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well-defined secondary structure in TFE and in TFE/water (90/10, v/v) with an alpha-helix encompassing residues 4-9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted alpha-helical conformation involving residues 4-8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the alpha-helical conformation even if separated from the rest of the molecule.     

DNA self-fitting: the double helix directs the geometry of its supramolecular assembly.

Groove-backbone interaction is a natural and biologically relevant mechanism for the specific assembly of B-DNA double helices. Crystal engineering and crystal packing analysis of oligonucleotides of different sizes and sequences reveal that the sequence-dependent self-fitting of B-DNA helices is a dominant constraint for their ordered assembly. It can override the other intermolecular interactions and impose the overall geometry of the packing. Analysis of experimental examples of architectural motifs formed by the geometric combination of self-fitted DNA segments leads to general rules for DNA assembly. Like a directing piece for a supramolecular 'construction set', the double helix imposes a limited number of geometric solutions. These basic architectural constraints could direct, in a codified manner, the formation of higher-order structures. DNA architectural motifs exhibit new structural and electrostatic properties which could have some implications for their molecular recognition by proteins acting on DNA.     

Chirality‐specific lift forces of helix under shear flows: Helix perpendicular to shear plane

Chiral objects in shear flow experience a chirality‐specific lift force. Shear flows past helices in a low Reynolds number regime were studied using slender‐body theory. The chirality‐specific lift forces in the vorticity direction experienced by helices are dominated by a set of helix geometry parameters: helix radius, pitch length, number of turns, and helix phase angle. Its analytical formula is given. The chirality‐specific forces are the physical reasons for the chiral separation of helices in shear flow. Our results are well supported by the latest experimental observations.     

Structure of the DNA-binding domain of the response regulator PhoP from Mycobacterium tuberculosis

The PhoP-PhoR two-component signaling system from Mycobacterium tuberculosis is essential for the virulence of the tubercle bacillus. The response regulator, PhoP, regulates expression of over 110 genes. In order to elucidate the regulatory mechanism of PhoP, we determined the crystal structure of its DNA-binding domain (PhoPC). PhoPC exhibits a typical fold of the winged helix-turn-helix subfamily of response regulators. The structure starts with a four-stranded antiparallel beta-sheet, followed by a three-helical bundle of alpha-helices, and then a C-terminal beta-hairpin, which together with a short beta-strand between the first and second helices forms a three-stranded antiparallel beta-sheet. Structural elements are packed through a hydrophobic core, with the first helix providing a scaffold for the rest of the domain to pack. The second and third helices and the long, flexible loop between them form the helix-turn-helix motif, with the third helix being the recognition helix. The C-terminal beta-hairpin turn forms the wing motif. The molecular surfaces around the recognition helix and the wing residues show strong positive electrostatic potential, consistent with their roles in DNA binding and nucleotide sequence recognition. The crystal packing of PhoPC gives a hexamer ring, with neighboring molecules interacting in a head-to-tail fashion. This packing interface suggests that PhoPC could bind DNA in a tandem association. However, this mode of DNA binding is likely to be nonspecific because the recognition helix is partially blocked and would be prevented from inserting into the major groove of DNA. Detailed structural analysis and implications with respect to DNA binding are discussed.     

Flexibility of DNA in 2:1 drug-DNA complexes--simultaneous binding of two DAPI molecules to DNA.

Simultaneous binding of two DAPI molecules in the minor groove of (dA)15.(dT)15 B-DNA helix has been simulated by molecular mechanics calculations. The energy minimised structure shows some novel features in relation to binding of DAPI molecules as well as the flexibility of the grooves of DNA helices. The minor groove of the helix expands locally considerably (to 15 angstroms) to accommodate the two DAPI molecules and is achieved by positive propeller twisting of base pairs at the binding site concomitant with small variations in the local nucleotide stereochemistry. The expansion also brings forth simultaneously a contraction in the width of the major groove spread over to a few phosphates. These findings demonstrate another facet of the flexible stereochemistry of DNA helices in which the local features are significantly altered without being propagated beyond a few base pairs, and with the rest of the regions retaining the normal structure. Both the DAPI molecules are engaged in specific hydrogen bonds with the bases and non specific interactions with phosphates. Stacking interactions of DAPI molecules between themselves as well as with sugar-phosphate backbone contribute to the stability of the complex. The studies provide a stereochemical support to the experimental findings that under high drug-DNA ratio DAPI could bind in the 2:1 ratio.     

Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove.     

The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix

The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.     

Open states in native polynucleotides. I. Hydrogen-exchange study of adenine-containing double helices.

Since protons that are buried and hydrogen-bonded within nucleic acid double helices exchange readily with solvent protons, it is evident that the native double helix must participate in some kind of reversible opening process. In hydrogen-exchange studies of a number of adenine-containing double helices, the chemical exchange pathways were worked out, and equilibrium and kinetic parameters of the dominant opening reactions were derived. These lead to a picture of the open state that may have implications for DNA recognition processes.     

Signatures of Protein-DNA Recognition in Free DNA Binding Sites

One obstacle to achieving complete understanding of the principles underlying sequence-dependent recognition of DNA is the paucity of structural data for DNA recognition sequences in their free (unbound) state. Here, we carried out crystallization screening of 50 DNA duplexes containing cognate protein binding sites and obtained new crystal structures of free DNA binding sites for three distinct modes of DNA recognition: anti-parallel β strands (MetR), helix-turn-helix motif + hinge helices (PurR), and zinc fingers (Zif268). Structural changes between free and protein-bound DNA are manifested differently in each case. The new DNA structures reveal that distinctive sequence-dependent DNA geometry dominates recognition by MetR, protein-induced bending of DNA dictates recognition by PurR, and deformability of DNA along the A-B continuum is important in recognition by Zif268. Together, our findings show that crystal structures of free DNA binding sites provide new information about the nature of protein-DNA interactions and thus lend insights towards a structural code for DNA recognition.