Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly ...conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.
Lipoplex preparations are heterogeneous mixtures of lipoplex particles of different structure. Because these structures determine the efficiency of genetic material delivery, it is important to ...characterize the distribution of particles of different types in lipoplex preparations with good statistics. We describe the application of flow fluorometry which allow producing such distributions (in terms of lipoplex particle size and composition) within minutes using basic flow cytometer (Pozharski and MacDonald, Anal Biochem 341:230-240, 2005).
We describe three applications of FRET technique to analyze structural and thermodynamic properties of cationic lipids and their complexes with DNA: (1) Lipid mixing assay to determine the degree to ...which individual vesicles undergo fusion upon complex formation. (2) DNA binding assay to obtain cationic lipid-DNA binding curves for thermodynamic analysis or binding stoichiometry characterization. (3) DNA spacing assay to determine changes in DNA packing in the multilamellar lipoplex particle.
Visualizing ligand molecules in twilight electron density Weichenberger, Christian X.; Pozharski, Edwin; Rupp, Bernhard
Acta crystallographica. Section F, Structural biology and crystallization communications,
February 2013, Letnik:
69, Številka:
2
Journal Article
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Three‐dimensional models of protein structures determined by X‐ray crystallography are based on the interpretation of experimentally derived electron‐density maps. The real‐space correlation ...coefficient (RSCC) provides an easily comprehensible, objective measure of the residue‐based fit of atom coordinates to electron density. Among protein structure models, protein–ligand complexes are of special interest, given their contribution to understanding the molecular underpinnings of biological activity and to drug design. For consumers of such models, it is not trivial to determine the degree to which ligand‐structure modelling is biased by subjective electron‐density interpretation. A standalone script, Twilight, is presented for the analysis, visualization and annotation of a pre‐filtered set of 2815 protein–ligand complexes deposited with the PDB as of 15 January 2012 with ligand RSCC values that are below a threshold of 0.6. It also provides simplified access to the visualization of any protein–ligand complex available from the PDB and annotated by the Uppsala Electron Density Server. The script runs on various platforms and is available for download at http://www.ruppweb.org/twilight/.
Helicobacter pylori NikR (HpNikR) is a nickel-dependent transcription factor that regulates multiple genes in the H. pylori pathogen. There are conflicting data regarding the locations of the Ni(II) ...sites and the role of Ni(II) coordination in DNA recognition. Herein, we report crystal structures of (i) the metal-binding domain (MBD) of HpNikR (3.08 Å) and (ii) a mutant, H74A (2.04 Å), designed to disrupt native Ni(II) coordination. In the MBD structure, four nickel ions are coordinated to two different types of nickel sites (4-coordinate, square planar, and 5/6-coordinate, square pyramidal/octahedral). In the H74A structure, all four nickel ions are coordinated to 4-coordinate square-planar sites. DNA-binding studies reveal tighter binding for target DNA sequences for holo-HpNikR compared with the affinities of Ni(II) reconstituted apo-HpNikR and H74A for these same DNA targets, supporting a role for Ni(II) coordination to 5/6 sites in DNA recognition. Small-angle X-ray scattering studies of holo-HpNikR and H74A reveal a high degree of conformational flexibility centered at the DNA-binding domains of H74A, which is consistent with disorder observed in the crystal structure of the protein. A model of DNA recognition by HpNikR is proposed in which Ni(II) coordination to specific sites in the MBD have a long-range effect on the flexibility of the DNA-binding domains and, consequently, the DNA recognition properties.
Structure-based drug design is underway to inhibit the S100B–p53 interaction as a strategy for treating malignant melanoma. X-ray crystallography was used here to characterize an interaction between ...Ca2+–S100B and TRTK-12, a target that binds to the p53-binding site on S100B. The structures of Ca2+–S100B (1.5-Å resolution) and S100B–Ca2+–TRTK-12 (2.0-Å resolution) determined here indicate that the S100B–Ca2+–TRTK-12 complex is dominated by an interaction between Trp7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca2+–S100B involving residues in helices 2 and 3 and loop 2. As with an S100B–Ca2+–p53 peptide complex, TRTK-12 binding to Ca2+–S100B was found to increase the protein's Ca2+-binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca2+ ligands and/or improved the Ca2+ coordination geometry of S100B. This possibility was ruled out when the structures of S100B–Ca2+–TRTK-12 and S100B–Ca2+ were compared and calcium ion coordination by the protein was found to be nearly identical in both EF-hand calcium-binding domains (RMSD=0.19). On the other hand, B-factors for residues in EF2 of Ca2+–S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR 15N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca2+–S100B. Such a loss of protein motion may also provide an explanation for how calcium-ion-binding affinity is increased upon binding a target. Lastly, it follows that any small-molecule inhibitor bound to Ca2+–S100B would also have to cause an increase in calcium-ion-binding affinity to be effective therapeutically inside a cell, so these data need to be considered in future drug design studies involving S100B.
A flow fluorometric analysis (using a commercial flow cytometer) of individual cationic lipoid–DNA complexes is presented. Such single lipoplex studies have the advantage of providing detailed ...characterization of heterogeneous ensembles of lipoplex preparations that cannot be obtained with methods that provide only population averages. Specifically, the composition (amount of lipoid and the lipoid–DNA ratio) was determined for statistically large ensembles (103–104 particles) under a variety of conditions, including DNA:lipoid mixing ratio, lipoid dispersion method (extruded, vortexed), DNA morphology (linear, supercoiled), and concentration. In addition, the kinetics of formation were assessed for several conditions. Under essentially all conditions, two distinct regimes were observed, and on the basis of present and past data, these were identified as (1) coexistence of multilamellar lipoplexes and DNA-coated vesicles and (2) highly fused multilamellar complexes. The former outcome is favored by excess of DNA, reduced vesicle size, linear DNA, high concentration, and short incubation times. Fused multilamellar complexes represent the structures of lipoplexes usually used for DNA transfection; these were formed by interaction and breakdown of DNA-coated vesicles. Because the composition of individual lipoplexes could be determined, it was possible to assess how much of the bulk sample heterogeneity originates within individual vesicles and how much is due to differences between lipoplexes.
As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca2+-loaded and Zn2+,Ca2+-loaded S100B were determined by X-ray crystallography at 2.15 Å (Rfree=0.266) and 1.85 Å ...(Rfree=0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca2+-loaded S100B and Zn2+,Ca2+-loaded S100B determined here (1.88 Å; Rfree=0.267). In the presence and absence of Zn2+, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (±Zn2+), and the second Pnt molecule was mapped to the dimer interface (site 2; ±Zn2+) and in a pocket near residues that define the Zn2+ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn2+ to Ca2+–S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt–Zn2+,Ca2+–S100B when compared to Pnt–Ca2+–S100B. That Pnt can adapt to this Zn2+-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca2+- and Ca2+,Zn2+-bound S100B.
The comparison of biomacromolecular crystal structures is traditionally based on the root‐mean‐square distance between corresponding atoms. This measure is sensitive to the presence of outliers, ...which inflate it disproportionately to their fraction. An alternative measure, the percentile‐based spread (p.b.s.), is proposed and is shown to represent the average variation in atomic positions more adequately. It is discussed in the context of isomorphous crystal structures, conformational changes and model ensembles generated by repetitive automated rebuilding.
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