Side Chain Carbonyl Research Articles

Regioselective reduction of 2-perfluoroalkanoylcyclohexane-1,3-diones and their enamino derivatives

Ionic hydrogenation of 2-perfluoroalkanoylcyclohexane-1,3-diones and their endocyclic enamino derivatives containing a secondary amino group by the action of triethylsilane in trifluoroacetic acid in the presence of a catalytic amount of lithium perchlorate involved regioselective reduction of the side-chain carbonyl group to hydroxy with formation of the corresponding hydroxy diketones and hydroxy amino ketones, respectively. Under analogous conditions endocyclic enamino derivatives possessing a tertiary amino group underwent deacylation to give enamino ketones.

Non-covalent interactions of alkali metal cations with singly charged tryptic peptides

The complexes formed by alkali metal cations (Cat(+) = Li(+), Na(+), K(+), Rb(+)) and singly charged tryptic peptides were investigated by combining results from the low-energy collision-induced dissociation (CID) and ion mobility experiments with molecular dynamics and density functional theory calculations. The structure and reactivity of [M + H + Cat](2+) tryptic peptides is greatly influenced by charge repulsion as well as the ability of the peptide to solvate charge points. Charge separation between fragment ions occurs upon dissociation, i.e. b ions tend to be alkali metal cationised while y ions are protonated, suggesting the location of the cation towards the peptide N-terminus. The low-energy dissociation channels were found to be strongly dependant on the cation size. Complexes containing smaller cations (Li(+) or Na(+)) dissociate predominantly by sequence-specific cleavages, whereas the main process for complexes containing larger cations (Rb(+)) is cation expulsion and formation of [M + H](+). The obtained structural data might suggest a relationship between the peptide primary structure and the nature of the cation coordination shell. Peptides with a significant number of side chain carbonyl oxygens provide good charge solvation without the need for involving peptide bond carbonyl groups and thus forming a tight globular structure. However, due to the lack of the conformational flexibility which would allow effective solvation of both charges (the cation and the proton) peptides with seven or less amino acids are unable to form sufficiently abundant [M + H + Cat](2+) ion. Finally, the fact that [M + H + Cat](2+) peptides dissociate similarly as [M + H](+) (via sequence-specific cleavages, however, with the additional formation of alkali metal cationised b ions) offers a way for generating the low-energy CID spectra of 'singly charged' tryptic peptides.

Carbonyl side-chain of catechol compounds is a key structure for the suppression of copper-associated oxidative DNA damage in vitro

Catechol is possibly carcinogenic to humans (International Agency for Research on Cancer, IARC). The key mechanism could include its oxidative DNA-damaging effect in combination with reductive–oxidative metals like Cu. We found that DNA damage was suppressed by introducing an α-carbonyl group to catechol at C4-position to produce carbonyl catechols. During the oxidative DNA-damaging process, catechols but not carbonyl catechols were oxidized to o-quinone; however, coexisting Cu(II) was reduced to Cu(I). Carbonyl catechols were possibly arrested at the oxidation step of semiquinones in the presence of Cu(II). Cu(I)-binding to DNA was stronger than Cu(II)-binding, on the basis of the circular dichroism spectral change. None of the carbonyl catechols induced such change, suggesting sequestration of Cu(I) from DNA. Solid-phase extraction experiments and spectrophotometric analyses showed the formation of semiquinone chelates with Cu(I). Thus, chelate formation could explain the suppression mechanism of the Cu–catechol-dependent DNA damage by terminating the reduction–oxidation cycle. Structural modifications such as introducing an α-carbonyl group to catechol at C4-position would contribute to reducing the risk and improving industrial and medical potentials of aromatic/phenolic compounds sustaining our daily lives.

Crystal Structure of the Ectodomain Complex of the CGRP Receptor, a Class-B GPCR, Reveals the Site of Drug Antagonism

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

ChemInform Abstract: Stereoselective Syntheses of Bridgehead Hydroxy Compounds via Reductive Cyclization Reactions Promoted by Samarium Diiodide.

Abstract Cycloalkanones appended with an α, β-unsaturated carbonyl side chain react with 2.25 equivalents of samarium diiodide to form bicyclic or tricyclic bridgehead hydroxy compounds stereoselectively in moderate yield.

Enantioselective One-Pot Rhodium-Catalyzed Cycloisomerization-Wittig Sequence to Chiral Functionalized 4-Alkyl 3-Alkylidene Tetrahydrofuran(on)es

Alkyl and (hetero)aryl alkyne allyl alcohols can readily be transformed into chiral 4-alkyl 3-alkylidene tetrahydrofurans and tetrahydrofuranones bearing α,β-unsaturated carbonyl side chains in a one-pot fashion via an enantioselective rhodium-catalyzed ­cycloisomerization-Wittig olefination sequence in good yields (54-86%).

Quantum Chemical Investigations on Intraresidue Carbonyl−Carbonyl Contacts in Aspartates of High-Resolution Protein Structures

The folding and stability of a polypeptide chain are due to many different and simultaneous noncovalent interactions. Recent studies have observed several novel and counterintuitive contacts in protein structures, and the nature of interactions due to such contacts is yet to be fully elucidated. We have identified carbonyl-carbonyl intraresidue contacts in 102 Asp residues from a data set of high-resolution protein structures. At the outset, it appears that such close approach of two carbonyl oxygen atoms is energetically not favorable. We have carried out ab initio quantum chemical calculations on 10 representative examples of self-contacting Asp residues from different regions of the Ramachandran map. Potential energy scan using three levels of theory (HF, B3LYP, and MP2) and two basis sets (6-31+G* and 6-31++G**) was performed by varying the side-chain dihedral angle chi(1) while keeping all other parameters corresponding to that observed in the protein structures. We also calculated interaction energies by considering the surrounding interacting residues and water molecules. Our results show that the energy difference between a self-contacting Asp residue from the crystal structures and the minimum energy conformations is about 10-15 kcal/mol. This small energy difference is compensated by its interactions with the surrounding residues and water molecules as observed in the interaction energy analysis. The results are independent of the levels of theory used. The contacting carbonyl-carbonyl groups adopt a sheared parallel motif orientation which helps to expose both the backbone and side-chain carbonyl oxygen atoms and enable them to participate in tertiary interactions. Natural bond orbital calculations indicate that carbonyl-carbonyl groups in self-contacting Asp residues interact through n --> pi* electron delocalization. The geometry analysis and nature of chemical interactions together explain the rationale for the existence of such Asp residues in protein structures and their importance in the protein stability.

The Functional Curli Amyloid Is Not Based on In-register Parallel β-Sheet Structure

The extracellular curli proteins of Enterobacteriaceae form fibrous structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-beta structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli structure is not based on an in-register parallel beta-sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a beta-helix-like structure but are not sufficient to establish such a structure definitively.

Design of HIV-1 protease inhibitors with pyrrolidinones and oxazolidinones as novel P1'-ligands to enhance backbone-binding interactions with protease: synthesis, biological evaluation, and protein-ligand X-ray studies.

Structure-based design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors are described. In an effort to enhance interactions with protease backbone atoms, we have incorporated stereochemically defined methyl-2-pyrrolidinone and methyl oxazolidinone as the P1'-ligands. These ligands are designed to interact with Gly-27' carbonyl and Arg-8 side chain in the S1'-subsite of the HIV protease. We have investigated the potential of these ligands in combination with our previously developed bis-tetrahydrofuran (bis-THF) and cyclopentanyltetrahydrofuran (Cp-THF) as the P2-ligands. Inhibitor 19b with a (R)-aminomethyl-2-pyrrolidinone and a Cp-THF was shown to be the most potent compound. This inhibitor maintained near full potency against multi-PI-resistant clinical HIV-1 variants. A high resolution protein-ligand X-ray crystal structure of 19b-bound HIV-1 protease revealed that the P1'-pyrrolidinone heterocycle and the P2-Cp-ligand are involved in several critical interactions with the backbone atoms in the S1' and S2 subsites of HIV-1 protease.

Resolving the Complications Induced by the Side Chain Carbonyl of Glutamine

IR spectroscopy is a widely used technique for structural studies of proteins which focuses on the amide I mode, an efficient marker of secondary structure. However, the use of IR for studying the glutamine based proteins associated with diseases such as Huntington's disease and Spinobulbar muscular atrophy is limited due to the additional complications introduced by the side chain carbonyl of glutamine. The two carbonyls will both vibrate in the amide I region, giving rise to multiple amide I modes; these must be resolved and unequivocally assigned if one is to understand the IR spectra of proteins containing glutamine. Furthermore, there is the opportunity for coupling between the two carbonyls through an electrostatic interaction known as transition dipole coupling (TDC). TDC causes a delocalization of the amide I vibrational wavefunction across oscillators that vibrate at similar frequencies (e.g. the backbone carbonyls of a protein or the two carbonyls of glutamine) and affects the frequency and transition strengths of the observed IR bands. TDC is dependent on the proximity and alignment of the carbonyls and thus is affected by the secondary structure of the protein as well as the side chain arrangement.Here we report a fundamental study of the IR spectra of glutamine that will ultimately guide the spectral assignment of the many amide I bands observed in the IR spectra of long stretches of polyglutamine residues (pQ32 and pQ43). We explore the hypothesis that the side chain of glutamine can form a hydrogen bond with the backbone, resulting in a cyclic conformation with a fixed alignment of the two carbonyls. The amidated form of glutamine has been studied at varying pH and temperature; data has been compared to that obtained for amidated alanine, amidated asparagine and non-amidated glutamine.

Synthesis of Pro-Xylane TM: A new biologically active C-glycoside in aqueous media

The scope and limitation of Lubineau’s reaction were evaluated for the synthesis of C-glycosides (compounds 1– 13). Further transformation of side chain carbonyl was also achieved (compounds 16– 23). Optimization of these two steps was investigated in xylose case. Some of the compounds were shown to stimulate sulfated glycosaminoglycans (GAGs) synthesis. Compound 20 (called Pro-Xylane TM) was identified as the best activator of GAGs biosynthesis. Pro-Xylane TM was developed using environmentally friendly conditions relevant to ‘Green-Chemistry’ principles and launched on the market in September 2006. This compound is the first example of ‘Green’ chemical used in cosmetic.

Structural and Functional Significance of Amino Acid Lysine192 (chymotrypsin numbering) in Factor XIa and Factor VIIa.

In blood coagulation serine proteases, amino acid 192 (chymotrypsin numbering) influences substrate and inhibitor specificity, and also forms a peptide bond with Gly193, a critical part of the protease oxyanion hole. Residue 192 is either Glu or Gln in several serine proteases including thrombin, factor (F) Xa, FIXa, and activated protein C. However, it is Lys in factor FXIa and FVIIa. Both FXIa and FVIIa/tissue factor (TF) share FIX as their substrate. In addition, FVIIa/TF activates FX and is inhibited by TF pathway inhibitor (TFPI), whereas FXIa is inhibited by diisopropylfluorophosphate (DFP), p-aminobenzamidine (p-AB), antithrombin and amyloid-b precursor protein Kunitz domain (AbPP). We investigated the importance of Lys192 in FXIa and FVIIa for substrate/inhibitor specificity and oxyanion hole formation. Recombinant FXIa was prepared with Pro (FXIaK192P), Glu (FXIaK192E) or Gln (FXIaK192Q) substitutions for Lys192. Rate constants for inhibition by DFP were similar for wild type FXIa (FXIaWT), FXIaK192E and FXIaK192Q, but 80-fold slower for FXIaK192P. Only FXIaK192P differed in binding to pAB compared to FXIaWT. These data indicate that Pro192 causes distortion of the oxyanion hole and S1 binding site. All mutants bound the FXIa substrate FIX normally. However, catalysis of FIX was impaired 400, 35, and 110-fold for FXIaK192P, FXIaK192E, and FXIaK192Q, respectively, while k2 for inhibition by antithrombin was reduced 160, 15 and 16-fold. FXIaK192P bound to AbPP with 800-fold decreased affinity; while FXIaK192E and FXIaK192Q bound with 6-10-fold higher affinity. Modeling studies indicate that there is loss of the normal interaction between Lys192 and the substrate/inhibitor P3' residue in interactions between FXIaK192E and FXIaK192Q and factor IX and antithrombin. The Ki for Glu and Gln mutant inhibition by AbPP is slightly better probably due to the ability of these two amino acids to make two H-bonds as compared to only one for Lys192. Lys192 makes H-bond with the carbonyl O of Gly12 in AbPP, whereas Glu and Gln at this position make H-bonds with the carbonyl O of Cys14 and Ala16 in AbPP. In FXIaK192P, the oxyanion hole is impaired due to a flip in the 192-193 peptide bond causing the amide N of Gly193 to point away from the oxyanion hole, resulting in a severe impairment of all catalytic functions. Thus a non-proline residue 192 is essential for proper oxyanion hole formation in serine proteases, and Lys192 in FXIa contributes significantly towards macromolecular substrate catalysis and inhibitor binding. In contrast to FXIaWT, the 192-193 peptide bond in FVIIaWT ± TF is already in a nonstandard conformation in which the amide N of Gly193 points away, and the carbonyl O of the Lys192 points into the oxyanion hole (Bajaj, et al., J. Biol. Chem. 281, 24873-24888, 2006). The H-bond between the amide N of Gly193 and the carbonyl side chain of Gln143, a residue unique to FVIIa, maintains this nonstandard conformation in FVIIa. Notably, upon substrate binding the 192-193 bond flips 180° such that the oxyanion hole is fully formed. Now the H-bond is formed between the amide N of Gly193 and the carbonyl oxygen of the transition state intermediate. For this to occur, the H-bond between the amide N of Gly193 and the carbonyl side chain of Gln143 must first be broken. Previous studies have shown that changing Lys192 to Glu or Gln in FVIIa severely impairs its biologic activity. However, the structural basis for this impairment is not understood. We performed molecular modeling studies which reveal that the side chain of the Glu192 carboxyl group or of the Gln192 carbonyl group makes strong H-bond with the side chain NH2 group of Gln143; this is not possible for Lys192. Thus in Glu192 or Gln192 mutant of FVIIa, it is energetically difficult to break the H-bond between the amide N of Gly193 and the carbonyl side chain of Gln143 because the H-bond involving the side chain of Glu192 or Gln192 and the Gln143 must be simultaneously broken. This provides a structural rational for the impaired function of FVIIaK192E or FVIIaK192Q. To test this concept further, we prepared FVIIa in which Lys192 was replaced with alanine. All properties of this alanine mutant were normal substantiating the proposed structural role of Lys192 in FVIIa. This structural role is unique to FVIIa among all known serine proteases, as is the presence of residue Gln143 that contributes to this phenomenon.

1,2,3-Triazole derivatives as new cannabinoid CB1 receptor antagonists

This letter reports the new entry of novel 1,2,3-triazole derivatives as CB1 receptor antagonists. The design, synthesis and biological evaluation of N1 and N2 substituted 1,2,3-trizoles are described. The N2 substituted, symmetrical 1,2,3-triazoles are more potent ligands than the unsymmetrical analogues. The in vitro activity of these triazoles is further improved by inserting a methylene group between the central core and the carbonyl side chain. The most potent antagonists prepared in this series (IC 50 < 20 nM) are the triazoles containing benzyl amides. These triazoles also show excellent selectivity between CB1 and CB2 receptors (IC 50 > 10 μM for CB2; CB2/CB1 > 1000).

Inhibitory Effect of Dehydrozingerone on Vascular Smooth Muscle Cell Function

Growth factor and oxidative stress-mediated migration and proliferation of vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of atherosclerosis. The objective of this study was to assess the ability of dehydrozingerone, a structural analog of curcumin, to inhibit PDGF-stimulated vascular functions in VSMCs. VSMCs isolated from adult rats were treated with dehydrozingerone (0 to 50 microM) before challenge with PDGF (10 ng/mL) and migration, proliferation, and collagen synthesis were assayed by transwell-migration, thymidine-, and L-proline-incorporation assays, respectively. Phosphorylation of PDGF-receptor (PDGFR) and Akt were assessed by Western blotting. Cellular protein tyrosine phosphatase (PTP) activity was determined by the extent of p-nitro-phenyl phosphate hydrolysis. Dehydrozingerone elicited a concentration-dependent inhibition of PDGF-stimulated VSMC migration, proliferation, collagen synthesis, and PDGF/H2O2-stimulated phosphorylation of PDGFR-beta and downstream Akt. Dehydrozingerone also inhibited H2O2-mediated oxidation of PTP. Dehydrozingerone is a potent inhibitor of growth factor/ H2O2-stimulated VSMC functions and may play a critical role in regulating these events after vascular injury. Inhibition of oxidation of cellular phosphatases may represent one of the mechanisms by which dehydrozingerone inhibits these VSMC functions. Inability of the structural analog isoeugenol to inhibit PDGF-signaling suggests that the carbonyl side chain may be necessary for activity.

Multiple-Timescale Dynamics of Side-Chain Carboxyl and Carbonyl Groups in Proteins by 13C Nuclear Spin Relaxation

Side-chain carboxyl and carbonyl groups play a major role in protein interactions and enzyme catalysis. A series of (13)C relaxation experiments is introduced to study the dynamics of carboxyl and carbonyl groups in protein side chains on both fast (sub-ns) and slower (micros-ms) time scales. This approach is illustrated on the protein calbindin D(9k). Fast dynamics features correlate with hydrogen- and ion-binding patterns. We also identify chemical dynamics on micros time scales in solvent-exposed carboxyl groups, most probably due to exchange between the carboxylate and carboxylic acid forms.

Advanced glycation end products serve as ligands for lectin‐like oxidized low‐density lipoprotein receptor‐1(LOX‐1): biochemical and binding characterizations assay

Advanced glycation end products (AGEs) are a class of complex heterogeneous compounds which accumulate with age and is known to be involved in the pathogenesis of several diseases from diabetes to atherosclerosis. AGEs serve as ligands for multiple receptors including scavenger receptor (SR-A), CD36, and SR-BIota. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays an important role in both atherosclerosis and is found to be an endothelial cell receptor for AGEs. To explore the binding characterization of AGEs to LOX-1, AGEs were prepared by three different reducing sugars (d-glucose, d-fructose, and d-ribose) and the biochemical characterization including, free amino groups, free amine content, fructosamine residues, carbonyl content, fluorescence, and absorbance were determined. The binding activity was determined by FITC labeled AGEs using Chinese hamster ovary-K1 cells stably transfected with human LOX-1 gene. The obtained AGEs showed significant differences in the extent of side chain modifications, carbonyl content, fluorescence, and absorption models. All of the AGEs showed specific and saturable binding to hLOX-1-CHO-K1 cells. Furthermore, dose-dependent binding processes were observed. However, the maximal cellular binding of AGEs differs between the sugars (glucose > ribose > fructose). In addition, oxidized low-density lipoprotein (ox-LDL) could significantly inhibit the binding of AGEs to LOX-1 with different inhibitory efficiency. LOX-1 serves as receptor for AGEs which may give some insight into the role of LOX-1 in the pathogenesis of diabetes and related disorders.

Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase

An extremely low-field signal (at approximately 18 p.p.m.) in the (1)H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theoretical calculations of chemical shift values were used in the interpretation of the experimental (1)H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 A resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 A) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor-acceptor distances of different types of hydrogen bonds. The short hydrogen-bond interactions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pK(a) values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins.

Thermal Stability of the K+Channel Tetramer: Cation Interactions and the Conserved Threonine Residue at the Innermost Site (S4) of the KcsA Selectivity Filter

The selectivity filter of most K+ channels contains a highly conserved Thr residue that uniquely forms the S4 binding site for K+ by dual coordination with the backbone carbonyl oxygen and side chain hydroxyl of the same residue. This study examines the effect of mutations of Thr75 in the S4 site of theKcsA K+ channel on the cation dependence of the thermal stability of the tetramer, a phenomenon that reflects the structural role of cations in the filter. Conservative mutations of Thr75 destabilize the tetramer and alter its temperature dependence. Replacement of Thr with Ala or Cys lowers the apparent affinity ofK+, Rb+, and Cs+ for tetramer stabilization by factors ranging from 4- to 14-fold. These same mutations lower the apparent affinity of Ba2+ by approximately 10(3)- or approximately 10(4)-fold for Ala and Cys substitution, respectively,consistent with the known preference of the S4 site for Ba2+. In contrast, substitution of Ala or Cys at T75 anomalously enhances the ability of Na+ to stabilize the tetramer, suggesting that the native Thr residue at S4 is important for ultrahigh K+/Na+ selectivity of K+ channel pores. Elevated temperature orCu2+ cation catalyzes formation of covalent dimers of the T75C mutant of KcsA via formation of disulfide bonds between Cys residues of adjacent subunits. Thiophilic cations such as Hg2+ and Ag+ specifically protect the T75C tetramer against heat-induced dimer formation, demonstrating the contribution of cation interactions to tetramer stability in a channel with a non-native S4 site engineered to bind foreign cations.

Crystallographic Studies on Multiple Conformational States of Active-site Loops in Pyrrolysyl-tRNA Synthetase

Pyrrolysine, a lysine derivative with a bulky pyrroline ring, is the “22nd” genetically encoded amino acid. In the present study, the carboxy-terminal catalytic fragment of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) was analyzed by X-ray crystallography and site-directed mutagenesis. The catalytic fragment ligated tRNA Pyl with pyrrolysine nearly as efficiently as the full-length PylRS. We determined the crystal structures of the PylRS catalytic fragment in the substrate-free, ATP analogue (AMPPNP)-bound, and AMPPNP/pyrrolysine-bound forms, and compared them with the previously-reported PylRS structures. The ordering loop and the motif-2 loop undergo conformational changes from the “open” states to the “closed” states upon AMPPNP binding. On the other hand, the β7-β8 hairpin exhibits multiple conformational states, the open, intermediate (β7-open/β8-open and β7-closed/β8-open), and closed states, which are not induced upon substrate binding. The PylRS structures with a docked tRNA suggest that the active-site pocket can accommodate the CCA terminus of tRNA when the motif-2 loop is in the closed state and the β7-β8 hairpin is in the open or intermediate state. The entrance of the active-site pocket is nearly closed in the closed state of the β7-β8 hairpin, which may protect the pyrrolysyladenylate intermediate in the absence of tRNA Pyl. Moreover, a structure-based mutational analysis revealed that hydrophobic residues in the amino acid-binding tunnel are important for accommodating the pyrrolysine side chain and that Asn346 is essential for anchoring the side-chain carbonyl and α-amino groups of pyrrolysine. In addition, a docking model of PylRS with tRNA was constructed based on the aspartyl-tRNA synthetase/tRNA structure, and was confirmed by a mutational analysis.

Self-contacts in Asx and Glx residues of high-resolution protein structures: Role of local environment and tertiary interactions

In protein structures, side-chains of asparagine and aspartic acid (Asx) and glutamine and glutamic acid (Glx) can approach their own backbone nitrogen or carbonyl group. We have systematically analyzed intra-residue contacts in Asx and Glx residues and their secondary structure preferences in two different datasets consisting of 500 and 1506 high-resolution structures. Intra-residue contact in an Asx/Glx residue between the heavy atoms of side-chain and main-chain functional groups of the same residue was investigated irrespective of whether such contacts are due to hydrogen bonding or not. Our search yielded 563 and 1462 cases of self-contacting Asx and Glx residues from the two datasets. Two important observations have been made in this analysis. First, self-contacts involving side-chain oxygen and backbone nitrogen atoms in majority of Asx residues are not due to hydrogen bonds. In the second instance, surprisingly, side-chain and backbone carbonyl oxygens of a significant number of Asx and Glx residues approach each other. For a wide-range of accessible surface areas, self-contacting residues are surrounded by less number of polar groups compared to all other Asx/Glx residues. In buried and partially buried regions, side-chain and main-chain functional groups of these residues together participate in simultaneous interactions with the available polar groups or water molecules. Asx/Glx residues with self-contacts are rarely observed in the middle of an α-helix or a β-strand. Asx/Glx side-chain having contact with its own backbone nitrogen shows different capping preferences compared to those having contact with its backbone oxygen. Examples of proteins with multiple self-contacting Asx/Glx residues are found. We speculate that mutation of a self-contacting residue in the buried or partially buried region of a protein will destabilize the structure. The results of this analysis will help in engineering protein structures and site-directed mutagenesis experiments.