Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: DNA binding specificity based on energetics of DNA kinking.

The catabolite activator protein (CAP) makes no direct contact with the consensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specificity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at position 7 form a T:A/G:C step, which is known to be associated with DNA flexibility. It has been proposed that specificity for T:A at position 6 is a consequence of formation of the DNA kink between positions 6 and 7, and of effects of the T:A(6)/G:C(7) step on the geometry of DNA kinking, or the energetics of DNA kinking. In this work, we determine crystallographic structures of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes containing T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this system does not involve differences in the geometry of DNA kinking but, rather, solely differences in the energetics of DNA kinking. We further infer that the main determinant of DNA conformation in this system is protein-DNA interaction, and not DNA sequence.

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking.

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), through an "indirect-readout" mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3' to the primary-kink site, G:C(7), through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C(7). Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A(6) to specificity for C:G(6), and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181-->Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A(6). The Glu181-->Asp substitution does not eliminate hydrogen-bond formation with G:C(7), and thus does not eliminate direct-readout-based specificity for G:C(7).

The crystal and molecular structure of a collagen-like peptide with a biologically relevant sequence.

A detailed description of the 2.0 A structure of the triple-helical peptide, (Pro-Hyp-Gly)(3)-Ile-Thr-Gly-Ala-Arg-Gly-Leu-Ala-Gly-Pro-Hyp-Gly-(Pro-Hyp-Gly)(3), denoted as T3-785, is presented. This peptide contains a biologically relevant sequence and was designed to model the imino acid-poor 785-796 region of human type III collagen just C-terminal to the matrix metalloproteinase cleavage site. The crystal structure of the T3-785 peptide demonstrates that sequence can influence local conformational changes in triple-helical structure, in terms of superhelical pitch, hydrogen bonding pattern, and hydration patterns. The novel packing arrangement displayed by the T3-785 structure, compared with those of collagen-like peptides with more imino acid-rich sequences indicates the sequence dependence of intermolecular assemblies in collagen as well. The observed synergy between the packing arrangements and the extended hydration network indicates that hydration of the triple helix is directly related to its association with other molecules.

Checking nucleic acid crystal structures.

The program SFCHECK [Vaguine et al. (1999), Acta Cryst. D55, 191-205] is used to survey the quality of the structure-factor data and the agreement of those data with the atomic coordinates in 105 nucleic acid crystal structures for which structure-factor amplitudes have been deposited in the Nucleic Acid Database [NDB; Berman et al. (1992), Biophys. J. 63, 751-759]. Nucleic acid structures present a particular challenge for structure-quality evaluations. The majority of these structures, and DNA molecules in particular, have been solved by molecular replacement of the double-helical motif, whose high degree of symmetry can lead to problems in positioning the molecule in the unit cell. In this paper, the overall quality of each structure was evaluated using parameters such as the R factor, the correlation coefficient and various atomic error estimates. In addition, each structure is characterized by the average values of several local quality indicators, which include the atomic displacement, the density correlation, the B factor and the density index. The latter parameter measures the relative electron-density level at the atomic position. In order to assess the quality of the model in specific regions, the same local quality indicators are also surveyed for individual groups of atoms in each structure. Several of the global quality indicators are found to vary linearly with resolution and less than a dozen structures are found to exhibit values significantly different from the mean for these indicators, showing that the quality of the nucleic acid structures tends to be rather uniform. Analysis of the mutual dependence of the values of different local quality indicators, computed for individual residues and atom groups, reveals that these indicators essentially complement each other and are not redundant with the B factor. Using several of these indicators, it was found that the atomic coordinates of the nucleic acid bases tend to be better defined than those of the backbone. One of the local indicators, the density index, is particularly useful in spotting regions of the model that fit poorly in the electron density. Using this parameter, the quality of crystallographic water positions in the analyzed structures was surveyed and it was found that a sizable fraction of these positions have poorly defined electron density and may therefore not be reliable. The possibility that cases of poorly positioned water molecules are symptomatic of more widespread problems with the structure as a whole is also raised.

Protein-RNA interactions: a structural analysis.

A detailed computational analysis of 32 protein-RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein-double-stranded DNA and protein-single-stranded DNA complexes. The interface properties of the protein-RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein-RNA and protein-DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein-RNA complexes, backbone contacts were more dominant in the protein-DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.

The PDB data uniformity project.

The Protein Data Bank (PDB; http://www.rcsb.org/pdb/) is the single worldwide archive of structural data of biological macromolecules. This paper describes the data uniformity project that is underway to address the inconsistency in PDB data.

The Protein Data Bank and the challenge of structural genomics.

The PDB has created systems for the processing, exchange, query, and distribution of data that will enable many aspects of high throughput structural genomics.

An overview of the structures of protein-DNA complexes.

On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes.

Staggered molecular packing in crystals of a collagen-like peptide with a single charged pair.

The crystal structure of the triple-helical peptide, (Pro-Hyp-Gly)(4)-Glu-Lys-Gly-(Pro-Hyp-Gly)(5) has been determined to 1.75 A resolution. This peptide was designed to examine the effect of a pair of adjacent, oppositely charged residues on collagen triple-helical conformation and intermolecular interactions. The molecular conformation (a 7(5) triple helix) and hydrogen bonding schemes are similar to those previously reported for collagen triple helices and provides a second instance of water mediated N--H . . . O==C interchain hydrogen bonds for the amide group of the residue following Gly. Although stereochemically capable of forming intramolecular or intermolecular ion pairs, the lysine and glutamic acid side-chains instead display direct interactions with carbonyl groups and hydroxyproline hydroxyl groups or interactions mediated by water molecules. Solution studies on the EKG peptide indicate stabilization at neutral pH values, where both Glu and Lys are ionized, but suggest that this occurs because of the effects of ionization on the individual residues, rather than ion pair formation. The EKG structure suggests a molecular mechanism for such stabilization through indirect hydrogen bonding. The molecular packing in the crystal includes an axial stagger between molecules, reminiscent of that observed in D-periodic collagen fibrils. The presence of a Glu-Lys-Gly triplet in the middle of the sequence appears to mediate this staggered molecular packing through its indirect water-mediated interactions with backbone C==O groups and side chains.

Integrin-collagen complex: a metal-glutamate handshake.

The recently determined crystal structure of the complex between an integrin I domain and a synthetic collagen peptide shows a collagen triple-helix engaged in specific macromolecular recognition and binding. This structure confirms a previously proposed binding mechanism for integrin I domains and has important implications for integrin activation and signalling.

The Protein Data Bank.

The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.

Sequence dependent conformational variations of collagen triple-helical structure.

The 2 A crystal structure reported here of the collagen-like model peptide, T3-785, provides the first visualization of how the sequence of collagen defines distinctive local conformational variations in triple-helical structure.

Protein-DNA interactions: A structural analysis.

A detailed analysis of the DNA-binding sites of 26 proteins is presented using data from the Nucleic Acid Database (NDB) and the Protein Data Bank (PDB). Chemical and physical properties of the protein-DNA interface, such as polarity, size, shape, and packing, were analysed. The DNA-binding sites shared common features, comprising many discontinuous sequence segments forming hydrophilic surfaces capable of direct and water-mediated hydrogen bonds. These interface sites were compared to those of protein-protein binding sites, revealing them to be more polar, with many more intermolecular hydrogen bonds and buried water molecules than the protein-protein interface sites. By looking at the number and positioning of protein residue-DNA base interactions in a series of interaction footprints, three modes of DNA binding were identified (single-headed, double-headed and enveloping). Six of the eight enzymes in the data set bound in the enveloping mode, with the protein presenting a large interface area effectively wrapped around the DNA.A comparison of structural parameters of the DNA revealed that some values for the bound DNA (including twist, slide and roll) were intermediate of those observed for the unbound B-DNA and A-DNA. The distortion of bound DNA was evaluated by calculating a root-mean-square deviation on fitting to a canonical B-DNA structure. Major distortions were commonly caused by specific kinks in the DNA sequence, some resulting in the overall bending of the helix. The helix bending affected the dimensions of the grooves in the DNA, allowing the binding of protein elements that would otherwise be unable to make contact. From this structural analysis a preliminary set of rules that govern the bending of the DNA in protein-DNA complexes, are proposed.

The past and future of structure databases.

For many years the primary source of information about biological macromolecules was the Protein Data Bank, which is a repository of files containing coordinates of biological macromolecules. Now a variety of databases has been developed in which structures are organized in different ways. Currently available are databases that classify molecules according to their overall structural characteristics or particular features of structures, as well as databases that focus on particular protein families in depth.

The crystal structure of an autoprocessed Ser221Cys-subtilisin E-propeptide complex at 2.0 A resolution.

We report here the crystallographic structure determination of an autoprocessed (Ser221Cys)-subtilisin E-propeptide complex at 2.0 A resolution. The subtilisin domain sequence has a single substitution (Ser221Cys) which has been shown to block the maturation process prior to degradation of the propeptide domain (77 residues) that acts as an intramolecular chaperon. This mutation, however, did not prevent the enzyme from cleaving its propeptide domain with a 60-80% efficiency. The current determination is the first example of a subtilisin E-propeptide complex which has been autoprocessed. A previous structure determination of a BPN'-prosegment complex has been reported in which the subtilisin domain was extensively mutated and a calcium binding loop was deleted. Further, in this earlier determination, the complex was formed by the addition of separately expressed propeptide domain. The structure determination reported here provides additional information about the nature of the interaction between the subtilisin and propeptide domains in this complex.

An analysis of the relationship between hydration and protein-DNA interactions.

Eleven protein-DNA crystal structures were analyzed to test the hypothesis that hydration sites predicted in the first hydration shell of DNA mark the positions where protein residues hydrogen-bond to DNA. For nine of those structures, protein atoms, which form hydrogen bonds to DNA bases, were found within 1.5 A of the predicted hydration positions in 86% of the interactions. The correspondence of the predicted hydration sites with the hydrogen-bonded protein side chains was significantly higher for bases inside the conserved DNA recognition sequences than outside those regions. In two CAP-DNA complexes, predicted base hydration sites correctly marked 71% (within 1.5 A) of protein atoms, which form hydrogen bonds to DNA bases. Phosphate hydration was compared to actual protein binding sites in one CAP-DNA complex with 78% marked contacts within 2.0 A. These data suggest that hydration sites mark the binding sites at protein-DNA interfaces.

Hydration of the phosphate group in double-helical DNA.

Water distributions around phosphate groups in 59 B-, A-, and Z-DNA crystal structures were analyzed. It is shown that the waters are concentrated in six hydration sites per phosphate and that the positions and occupancies of these sites are dependent on the conformation and type of nucleotide. The patterns of hydration that are characteristic of the backbone of the three DNA helical types can be attributed in part to the interactions of these hydration sites.

Maximum A posteriori classification of DNA structure from sequence information.

We introduce an algorithm, LLLAMA, which combines simple pattern recognizers into a general method for estimating the entropy of a sequence. Each pattern recognizer exploits a partial match between subsequences to build a model of the sequence. Since the primary features of interest in biological sequence domains are subsequences with small variations in exact composition, LLLAMA is particularly suited to such domains. We describe two methods, LLLAMA-length and LLLAMA-alone, which use this entropy estimate to perform maximum a posteriori classification. We apply these methods to several problems in three-dimensional structure classification of short DNA sequences. The results include a surprisingly low 3.6% error rate in predicting helical conformation of oligonucleotides. We compare our results to those obtained using more traditional methods for automated generation of classifiers.