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.

Absent, small, or homeotic disc1 (Ash1) is a trithorax group histone methyltransferase that is involved in gene activation. Although there are many known histone methyltransferases, their regulatory mechanisms are poorly understood. Here, we present the crystal structure of the human ASH1L catalytic domain, showing its substrate binding pocket blocked by a loop from the post-SET domain. In this configuration, the loop limits substrate access to the active site. Mutagenesis of the loop stimulates ASH1L histone methyltransferase activity, suggesting that ASH1L activity may be regulated through the loop from the post-SET domain. In addition, we show that human ASH1L specifically methylates histone H3 Lys-36. Our data implicate that there may be a regulatory mechanism of ASH1L histone methyltransferases.Nucleosomes, the fundamental unit of the highly ordered chromatin structure, are composed of DNA wrapped around histone octamers. Histones have N-terminal tails that are exposed on the outside of nucleosomes. These tails are subjected to several post-translational modifications, including acetylation, phosphorylation, ubiquitination, sumoylation, and methylation (1).The site-specific methylation of histone lysine residues is important for the epigenetic control of gene expression. These marks serve to regulate epigenetically the organization of chromatin structure and to recruit other chromatin modifiers (2, 3). Methylation can occur at multiple lysine residues, including lysines 4, 9, 27, 36, and 79 of histone H3 and lysine 20 of histone H4. Absent, small, or homeotic disc1 (Ash1) is a member of the trithorax group proteins, which are essential for epigenetic gene activation (4,5). Previous studies have shown that Drosophila Ash1 activates homeotic gene ultrabithorax expression in imaginal discs of the third leg (6) and interacts with trithorax to regulate and maintain ultrabithorax expression (7). It has also been reported that the mammalian homolog of Ash1, ASH1L, is a histone methyltransferase (HMTase) 2 that is associated with transcribed regions of active genes (8, 9). ASH1L has several domains, including an associated with SET domain (AWS), a SET domain, a post-SET domain, a bromodomain, a bromoadjacent homology domain (BAH), and a plant homeodomain finger (SMART database (10)). The SET domain in HMTases is responsible for catalyzing the formation of monomethylated, dimethylated, and trimethylated lysine, establishing an additional complex system with respect to methylated lysine recognition in signaling (11). Several crystal structures of SET domain proteins have been solved, and they revealed that the SET domain forms a knot-like structure that constitutes the active site of HMTases (12). Notably, ASH1L contains a SET domain in the middle of the protein, whereas other proteins possess a SET domain at the C terminus. Drosophila Ash1 has been previously shown to affect H3K4 methylation levels genetically and to methylate H3K4, H3K9, and H4K20 in in vitro assays (8, 13). The Tanaka group (14) has found H3K36 ...

The series of events that occur in the catalytic cycle of matrix metalloproteinases were modeled on the basis of X‐ray crystal structures of the active, uninhibited enzymes and of the same enzymes following the hydrolysis of a peptide substrate. After the peptide bond has been broken, both peptide fragments remain bound to the protein initially (see structure of the active‐site cavity of the enzyme MMP‐12 immediately after substrate hydrolysis).

By solving high-resolution crystal structures of a large number (14 in this case) of adducts of matrix metalloproteinase 12 (MMP12) with strong, nanomolar, inhibitors all derived from a single ligand scaffold, it is shown that the energetics of the ligand-protein interactions can be accounted for directly from the structures to a level of detail that allows us to rationalize for the differential binding affinity between pairs of closely related ligands. In each case, variations in binding affinities can be traced back to slight improvements or worsening of specific interactions with the protein of one or more ligand atoms. Isothermal calorimetry measurements show that the binding of this class of MMP inhibitors is largely enthalpy driven, but a favorable entropic contribution is always present. The binding enthalpy of acetohydroxamic acid (AHA), the prototype zinc-binding group in MMP drug discovery, has been also accurately measured. In principle, this research permits the planning of either improved inhibitors, or inhibitors with improved selectivity for one or another MMP. The present analysis is applicable to any drug target for which structural information on adducts with a series of homologous ligands can be obtained, while structural information obtained from in silico docking is probably not accurate enough for this type of study.

The heterodimeric structure of the MST1 and RASSF5 SARAH domains is presented. A comparison of homodimeric and heterodimeric interactions provides a structural basis for the preferential association of the SARAH heterodimer.

A combination of in silico tools and experimental NMR data is proposed for relatively fast determination of protein-ligand structural models and demonstrated from known inhibitors of matrix metalloproteinases (MMP). The 15N 1H heteronuclear single quantum coherence (HSQC) spectral assignment and the 3D structure, either X-ray or NMR, are needed. In this method, the HSQC spectrum with or without the ligand is used to determine the interaction region of the ligand. Docking calculations are then performed to obtain a set of structural models. From the latter, the nuclear Overhauser effects (NOEs) between the ligand and the protein can be predicted. Guided by these predictions, a number of NOEs can be detected and assigned through a HSQC NOESY experiment. These data are used as structural restraints to reject/refine the initial structural models through further in silico work. For a test protein (MMP-12, human macrophage metalloelastase), a final structure of a protein-ligand adduct was obtained that matches well with the full structural determination. A number of structural predictions were then made for adducts of a similar protein (MMP-1, human fibroblast collagenase) with the same and different ligands. The quality of the final results depended on the type and number of experimental NOEs, but in all cases, a well-defined ligand conformation in the protein binding site was obtained. This protocol is proposed as a viable alternative to the many approaches described in the literature.

Die Abfolge von Ereignissen, die im Katalysezyklus von Matrix‐Metalloproteinasen eintreten, wurde auf der Basis von Kristallstrukturen der aktiven, nicht inhibierten Enzyme und derselben Enzyme nach Hydrolyse eines Peptidsubstrats modelliert. Nach dem Bruch der Peptidbindung bleiben beide Peptidfragmente zunächst an das Protein gebunden (siehe Struktur der Kavität am aktiven Zentrum des Enzyms MMP‐12 direkt nach der Substrathydrolyse).

Data transfer. The solid‐state proton‐driven spin diffusion (PDSD) and J‐decoupled PDSD NMR spectra of the microcrystalline catalytic domain of matrix metalloproteinase 12 (MMP‐12, 17 kDa) have been recorded. It is shown that such spectra can be largely assigned in a few days by using the available liquid‐state assignment and validated with an independent sequential assignment based on 3D NCACX and NCOCX PDSD experiments. This demonstrates how quickly the liquid‐state assignment of comparably large protein can be transferred to the solid state.