[Show abstract][Hide abstract] ABSTRACT: Over the years, a great deal of effort has been focused on the design and synthesis of potent, linear peptide inhibitors targeting the polo-like kinase 1 (Plk1), which is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its intracellular anchoring sites via its polo-box domain, and inhibiting the Plk1 polo-box domain has been considered as an approach to circumvent the specificity problems associated with inhibiting the conserved adenosine triphosphate-binding pocket. The polo-box domain consists of two different binding regions, such as the unique, broader pyrrolidine-binding pocket and the conserved, narrow, Tyr-rich hydrophobic channel, among the three Plk polo-box domains (Plks 1-3), respectively. Therefore, the studies that provide insights into the binding nature of the unique, broader pyrrolidine-binding pocket might lead to the development of selective Plk1-inhibitory compounds.
In an attempt to retain the monospecificity by targeting the unique, broader pyrrolidine-binding pocket, here, for the first time, a systematic approach was undertaken to examine the structure-activity relationship of N-terminal-truncated PLHSpTM derivatives, to apply a site-directed ligand approach using bulky aromatic and non-aromatic systems, and to characterize the binding nature of these analogues using X-ray crystallographic studies. We have identified a new mode of binding interactions, having improved binding affinity and retaining the Plk1 polo-box domain specificity, at the pyrrolidine-binding pocket. Furthermore, our data revealed that the pyrrolidine-binding pocket was very specific to recognize a short and bulky hydrophobic ligand like adamantane, whereas the Tyr-rich hydrophobic channel was specific with lengthy and small hydrophobic groups.
The progress made using our site-directed ligands validated this approach to specifically direct the ligand into the unique pyrrolidine-binding region, and it extends the applicability of the strategy for discovering selective protein-protein interaction inhibitors.
PLoS ONE 11/2013; 8(11):e80043. DOI:10.1371/journal.pone.0080043 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The meso isomer of diaminopimelate (meso-DAP) is a biosynthetic precursor of L-lysine in bacteria and plants, and is a key component of the peptidoglycan layer in the cell walls of Gram-negative and some Gram-positive bacteria. Diaminopimelate epimerase (DapF) is a pyridoxal-5'-phosphate-independent racemase which catalyses the interconversion of (6S,2S)-2,6-diaminopimelic acid (LL-DAP) and meso-DAP. In this study, DapF from Acinetobacter baumannii was overexpressed in Escherichia coli strain SoluBL21, purified and crystallized using a vapour-diffusion method. A native crystal diffracted to a resolution of 1.9 Å and belonged to space group P3(1) or P3(2), with unit-cell parameters a = b = 74.91, c = 113.35 Å, α = β = 90, γ = 120°. There were two molecules in the asymmetric unit.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 01/2013; 69(Pt 1):42-4. DOI:10.1107/S1744309112048506 · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peptidoglycan-associated lipoprotein (Pal) is one component of the Tol-Pal system that is involved in maintaining the integrity and stability of the outer membrane. The C-terminal OmpA-like domain of Pal interacts noncovalently with peptidoglycan. In this study, the OmpA-like domain of Pal from Acinetobacter baumannii was overexpressed in Escherichia coli strain BL21 (DE3), purified and crystallized using the vapour-diffusion method. A native crystal diffracted to 1.4 Å resolution and belonged to space group P6(1) or P6(5), with unit-cell parameters a = b = 72.58, c = 44.65 Å, a calculated Matthews coefficient of 2.64 Å(3) Da(-1) and one molecule per asymmetric unit.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2012; 68(Pt 11):1351-1353. DOI:10.1107/S1744309112038924 · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HP0902 from Helicobacter pylori 26695 belongs to the cupin superfamily of proteins, which encompasses proteins with a great diversity in function. In this work, two types of recombinant HP0902 protein were crystallized: one with an N-terminal His(6) tag ((H6)HP0902) and the other with a C-terminal His(6) tag (HP0902(H6)). The (H6)HP0902 crystal diffracted to 1.40 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.5, b = 78.6, c = 41.4 Å. The HP0902(H6) crystal belonged to space group P4(3)2(1)2 or P4(1)2(1)2 and diffracted to 2.5 Å resolution, with unit-cell parameters a = b = 50.4, c = 142.0 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2011; 67(Pt 12):1619-22. DOI:10.1107/S1744309111039315 · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal β-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2(1), with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, β = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å(3) Da(-1).
Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2011; 67(Pt 12):1531-3. DOI:10.1107/S1744309111038401 · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The outer membrane protein A (OmpA) plays important roles in anchoring of the outer membrane to the bacterial cell wall. The C-terminal periplasmic domain of OmpA (OmpA-like domain) associates with the peptidoglycan (PGN) layer noncovalently. However, there is a paucity of information on the structural aspects of the mechanism of PGN recognition by OmpA-like domains. To elucidate this molecular recognition process, we solved the high-resolution crystal structure of an OmpA-like domain from Acinetobacter baumannii bound to diaminopimelate (DAP), a unique bacterial amino acid from the PGN. The structure clearly illustrates that two absolutely conserved Asp271 and Arg286 residues are the key to the binding to DAP of PGN. Identification of DAP as the central anchoring site of PGN to OmpA is further supported by isothermal titration calorimetry and a pulldown assay with PGN. An NMR-based computational model for complexation between the PGN and OmpA emerged, and this model is validated by determining the crystal structure in complex with a synthetic PGN fragment. These structural data provide a detailed glimpse of how the anchoring of OmpA to the cell wall of gram-negative bacteria takes place in a DAP-dependent manner.
The FASEB Journal 09/2011; 26(1):219-28. DOI:10.1096/fj.11-188425 · 5.48 Impact Factor