Human insulin polymorphism upon ligand binding and pH variation: the case of 4-ethylresorcinol.

This study focuses on the effects of the organic ligand 4-ethylresorcinol on the crystal structure of human insulin using powder X-ray crystallography. For this purpose, systematic crystallization experiments have been conducted in the presence of the organic ligand and zinc ions within the pH range 4.50-8.20, while observing crystallization behaviour around the isoelectric point of insulin. High-throughput crystal screening was performed using a laboratory X-ray diffraction system. The most representative samples were selected for synchrotron X-ray diffraction measurements, which took place at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). Four different crystalline polymorphs have been identified. Among these, two new phases with monoclinic symmetry have been found, which are targets for the future development of microcrystalline insulin drugs.

Novel crystalline phase and first-order phase transitions of human insulin complexed with two distinct phenol derivatives.

The primary focus of the present work is the study of the effects that two ligands and the crystallization pH have on the crystalline forms of human insulin. For this purpose, human insulin (HI) was co-crystallized with two distinct phenolic derivatives: the organic ligands meta-cresol (m-cresol) and 4-nitrophenol. The formation of polycrystalline precipitates was then followed by means of structural characterization of the individual specimens in terms of unit-cell symmetry and parameters. In both cases, two different polymorphs were identified via X-ray powder diffraction measurements, the first of hexagonal symmetry (R3 space group) at higher pH values and the second of monoclinic symmetry (space group P21) with unit-cell parameters a = 87.4282 (5), b = 70.5020 (3), c = 48.3180 (4) Å, ß = 106.8958 (4)°, the latter of which to our knowledge has never been observed before.

The 2.2 A structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism.

The rRNA methyltransferase ErmC' transfers methyl groups from S -adenosyl-l-methionine to atom N6 of an adenine base within the peptidyltransferase loop of 23 S rRNA, thus conferring antibiotic resistance against a number of macrolide antibiotics. The crystal structures of ErmC' and of its complexes with the cofactor S -adenosyl-l-methionine, the reaction product S-adenosyl-l-homocysteine and the methyltransferase inhibitor Sinefungin, respectively, show that the enzyme undergoes small conformational changes upon ligand binding. Overall, the ligand molecules bind to the protein in a similar mode as observed for other methyltransferases. Small differences between the binding of the amino acid parts of the different ligands are correlated with differences in their chemical structure. A model for the transition-state based on the atomic details of the active site is consistent with a one-step methyl-transfer mechanism and might serve as a first step towards the design of potent Erm inhibitors.

M.TaqI: possible catalysis via cation-pi interactions in N-specific DNA methyltransferases.

The adenine-specific DNA methyltransferase M.TaqI transfers a methyl group from S-adenosylmethionine to N6 of the adenine residue in the DNA sequence 5'-TCGA-3'. In the crystal structure of M.TaqI in complex with S-adenosylmethionine the enzyme is folded into two domains: An N-terminal catalytic domain, whose fold is conserved among S-adenosyl-methionine dependent methyltransferases, and a DNA recognition domain which possesses a unique fold. In the active site, two aromatic residues, Tyr 108 and Phe 196, are postulated to bind the flipped-out target DNA adenine which becomes methylated. By lowering the energy of the positively charged transition state via cationic-pi interactions, these two residues probably hold a key role in catalysis.

Crystal structure of ErmC', an rRNA methyltransferase which mediates antibiotic resistance in bacteria.

The prevalent mechanism of bacterial resistance to erythromycin and other antibiotics of the macrolide-lincosamide-streptogramin B group (MLS) is methylation of the 23S rRNA component of the 50S subunit in bacterial ribosomes. This sequence-specific methylation is catalyzed by the Erm group of methyltransferases (MTases). They are found in several strains of pathogenic bacteria, and ErmC is the most studied member of this class. The crystal structure of ErmC' (a naturally occurring variant of ErmC) from Bacillus subtilis has been determined at 3.0 A resolution by multiple anomalous diffraction phasing methods. The structure consists of a conserved alpha/beta amino-terminal domain which binds the cofactor S-adenosyl-l-methionine (SAM), followed by a smaller, alpha-helical RNA-recognition domain. The beta-sheet structure of the SAM-binding domain is well-conserved between the DNA, RNA, and small-molecule MTases. However, the C-terminal nucleic acid binding domain differs from the DNA-binding domains of other MTases and is unlike any previously reported RNA-recognition fold. A large, positively charged, concave surface is found at the interface of the N- and C-terminal domains and is proposed to form part of the protein-RNA interaction surface. ErmC' exhibits the conserved structural motifs previously found in the SAM-binding domain of other methyltransferases. A model of SAM bound to ErmC' is presented which is consistent with the motif conservation among MTases.

Differential binding of S-adenosylmethionine S-adenosylhomocysteine and Sinefungin to the adenine-specific DNA methyltransferase M.TaqI.

The crystal structures of the binary complexes of the DNA methyltransferase M.TaqI with the inhibitor Sinefungin and the reaction product S-adenosyl-L-homocysteine were determined, both at 2.6 A resolution. Structural comparison of these binary complexes with the complex formed by M.TaqI and the cofactor S-adenosyl-L-methionine suggests that the key element for molecular recognition of these ligands is the binding of their adenosine part in a pocket, and discrimination between cofactor, reaction product and inhibitor is mediated by different conformations of these molecules; the methionine part of S-adenosyl-L-methionine is located in the binding cleft, whereas the amino acid moieties of Sinefungin and S-adenosyl-L-homocysteine are in a different orientation and interact with the active site amino acid residues 105NPPY108. Dissociation constants for the complexes of M.TaqI with the three ligands were determined spectrofluorometrically. Sinefungin binds more strongly than S-adenosyl-L-homocysteine or S-adenosyl-L-methionine, with KD=0.34 microM, 2.4 microM and 2.0 microM, respectively.

A model for DNA binding and enzyme action derived from crystallographic studies of the TaqI N6-adenine-methyltransferase.

The crystal structures of the DNA-N6-adenine-methyltransferase M.TaqI, in complexes with the cofactor S-adenosyl-L-methionine (AdoMet) and the competitive inhibitor sinefungin (Sf) show identical folding of the polypeptide chains into two domains. The N-terminal domain carries the cofactor-binding site, the C-terminal domain is thought to be implicated in sequence-specific DNA binding. Model building of the M.TaqI-DNA complex suggests that the adenine to be methylated swings out of the double helix as found previously in the cytosine-C5-MTase HhaI DNA co-crystal structure. A torsion of the methionine moiety of the cofactor is required to bring the methyl group within reach of the swung-out base and allow methyl group transfer.

Universal catalytic domain structure of AdoMet-dependent methyltransferases.

The DNA methyltransferases, M.HhaI and M.TaqI, and catechol O-methyl-transferase (COMT) catalyze the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to carbon-5 of cytosine, to nitrogen-6 of adenine, and to a hydroxyl group of catechol, respectively. The catalytic domains of the bilobal proteins, M.HhaI and M.TaqI, and the entire single domain of COMT have similar folding with an alpha/beta structure containing a mixed central beta-sheet. The functional residues are located in equivalent regions at the carboxyl ends of the parallel beta-strands. The cofactor binding sites are almost identical and the essential catalytic amino acids coincide. The comparable protein folding and the existence of equivalent amino acids in similar secondary and tertiary positions indicate that many (if not all) AdoMet-dependent methyltransferases have a common catalytic domain structure. This permits tertiary structure prediction of other DNA, RNA, protein, and small-molecule AdoMet-dependent methyltransferases from their amino acid sequences.

Three-dimensional structure of the adenine-specific DNA methyltransferase M.Taq I in complex with the cofactor S-adenosylmethionine.

The Thermus aquaticus DNA methyltransferase M.Taq I (EC 2.1.1.72) methylates N6 of adenine in the specific double-helical DNA sequence TCGA by transfer of --CH3 from the cofactor S-adenosyl-L-methionine. The x-ray crystal structure at 2.4-A resolution of this enzyme in complex with S-adenosylmethionine shows alpha/beta folding of the polypeptide into two domains of about equal size. They are arranged in the form of a C with a wide cleft suitable to accommodate the DNA substrate. The N-terminal domain is dominated by a nine-stranded beta-sheet; it contains the two conserved segments typical for N-methyltransferases which form a pocket for cofactor binding. The C-terminal domain is formed by four small beta-sheets and alpha-helices. The three-dimensional folding of M.Taq I is similar to that of the cytosine-specific Hha I methyltransferase, where the large beta-sheet in the N-terminal domain contains all conserved segments and the enzymatically functional parts, and the smaller C-terminal domain is less structured.

X-ray crystallographic and calorimetric studies of the effects of the mutation Trp59-->Tyr in ribonuclease T1.

Two mutants of ribonuclease T1 (RNaseT1), [59-tyrosine]ribonuclease T1 (W59Y) and [45-tryptophan,59-tyrosine]ribonuclease T1 (Y45W/W59Y) possess between 150% and 190% wild-type activity. They have been crystallised as complexes of the inhibitor 2'-guanylic acid and analysed by X-ray diffraction at resolutions of 0.23 nm and 0.24 nm, respectively. The space group for both is monoclinic, P2(1), with two molecules/asymmetric unit, W59Y: a = 4.934 nm, b = 4.820 nm, c = 4.025 nm, beta = 90.29 degrees. Y45W/W59Y: a = 4.915 nm, b = 4.815 nm, c = 4.015 nm, beta = 90.35 degrees. Compared to wild-type RNaseT1 in complex with 2'-guanylic acid (2'GMP) both mutant inhibitor complexes indicate that the replacement of Trp59 by Tyr leads to a 0.04-nm inward shift of the single alpha-helix and to significant differences in the active-site geometry, inhibitor conformation and inhibitor binding. Calorimetric studies of a range of mutants [24-tryptophan]ribonuclease T1 (Y24W), [42-tryptophan]ribonuclease T1 (Y42W), [45-tryptophan]ribonuclease T1 (Y45W), [92-alanine]ribonuclease T1 (H92A) and [92-threonine]ribonuclease T1 (H92T) with and without the further mutation Trp59-->Tyr showed that mutant proteins for which Trp59 is replaced by Tyr exhibit slightly decreased thermal stability.