Enzyme function is often dependent on fluctuations between inactive and active structural ensembles. Adenylate kinase isolated from Escherichia coli (AK e ) is a small phosphotransfer enzyme in which interconversion between inactive (open) and active (closed) conformations is rate limiting for catalysis. AK e has a modular three-dimensional architecture with two flexible substrate-binding domains that interact with the substrates AmP, ADP and ATP. Here, we show by using a combination of biophysical and mutagenic approaches that the interconversion between open and closed states of the ATP-binding subdomain involves partial subdomain unfolding/refolding in an otherwise folded enzyme. These results provide a novel and, possibly general, molecular mechanism for the switch between open and closed conformations in AK e .
Four beta-linked glucobioses selectively (13)C labeled at C1' or C2' have been prepared. The inter-residue coupling constants, J(CH), and J(CC), have been determined and related to the solution conformations of the disaccharides using Karplus-type relationships. Relying only on the experimental coupling constants, glycosidic linkage conformation in methyl alpha-sophoroside (methyl 2-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside), methyl alpha-laminarabioside (methyl 3-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside), and methyl alpha-cellobioside (methyl 4-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside) were found to be close to those observed in the solid state (39 degrees < phi(H) < 41 degrees , -24 degrees < psi(H) < -36 degrees ). The laminarabioside and cellobioside were found to have conformations that accommodate an intramolecular hydrogen bond to O5' that is observed in the solid state. In all compounds, the exocyclic hydroxymethyl groups retain a conformation close to that observed in unsubstituted glucose (gt/gg 1:1). Methyl alpha-gentiobioside (methyl 6-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside) shows greater flexibility at the psi-torsion than the other disaccharides, but the population distribution around the C5-C6 bond is essentially unaffected by substitution. None of the O2' hydroxyl groups of the beta-D-glucopyranosyl residues in any of the disaccharides appear to be involved in inter-residue hydrogen bonding since (1)JCH, (1)JCC, and (2)JCH values sensitive to C2'-O2' rotamer distribution remain close to those observed in methyl beta-D-glucopyranoside.
Conformational change is regulating the biological activity of a large number of proteins and enzymes. Efforts in structural biology have provided molecular descriptions of the interactions that stabilize the stable ground states on the reaction trajectories during conformational change. Less is known about equilibrium thermodynamic stabilities of the polypeptide segments that participate in structural changes and whether the stabilities are relevant for the reaction pathway. Adenylate kinase (Adk) is composed of three subdomains: CORE, ATPlid, and AMPbd. ATPlid and AMPbd are flexible nucleotide binding subdomains where large-scale conformational changes are directly coupled to catalytic activity. In this report, the equilibrium thermodynamic stabilities of Adk from both mesophilic and hyperthermophilic bacteria were investigated using solution state NMR spectroscopy together with protein engineering experiments. Equilibrium hydrogen to deuterium exchange experiments indicate that the flexible subdomains are of significantly lower thermodynamic stability compared to the CORE subdomain. Using site-directed mutagenesis, parts of ATPlid and AMPbd could be selectively unfolded as a result of perturbation of hydrophobic clusters located in these respective subdomains. Analysis of the perturbed Adk variants using NMR spin relaxation and C(alpha) chemical shifts shows that the CORE subdomain can fold independently of ATPlid and AMPbd; consequently, folding of the two flexible subdomains occurs independently of each other. Based on the experimental results it is apparent that the flexible subdomains fold into their native structure in a noncooperative manner with respect to the CORE subdomain. These results are discussed in light of the catalytically relevant conformational change of ATPlid and AMPbd.
The description of the reorientational dynamics of flexible molecules is a challenging task, in particular when the rates of internal and global motions are comparable. The commonly used simple mode-decoupling models are based on the assumption of statistical independence between these motions. This assumption is not valid when the time scale separation between their rates is small, a situation that was found to arise in oligosaccharides in the context of certain internal motions. To make possible the interpretation of NMR spin relaxation data from such molecules, we developed a comprehensive approach generally applicable to flexible rotators with one internal degree of freedom. This approach integrates a stochastic description of coupled global tumbling and internal torsional motion, quantum chemical calculations of the local potential and the local geometry at the site of the restricted torsion, and hydrodynamics-based calculations of the diffusive properties. The method is applied to the disaccharide beta-D-Glcp-(1-->6)-alpha-D-[6-(13)C]-Manp-OMe dissolved in a DMSO-d(6)/D(2)O cryosolvent. The experimental NMR relaxation parameters, associated with the (13)CH(2) probe residing at the glycosidic linkage, include (13)C T(1) and T(2) and (13)C-{(1)H} nuclear Overhauser enhancement (NOE) as well as longitudinal and transverse dipole-dipole cross-correlated relaxation rates, acquired in the temperature range of 253-293 K. These data are predicted successfully by the new theory with only the H-C-H angle allowed to vary. Previous attempts to fit these data using mode-decoupling models failed.
Significance Leukotriene (LT) A 4 hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes biosynthesis of the proinflammatory mediator, LTB 4 , implicated in chronic inflammatory diseases. Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as the enzyme’s endogenous peptidase substrate. Pro-Gly-Pro is cleaved and inactivated by LTA4H, suggesting that LTA4H plays a role in both the initiation and the resolution phase of inflammation. Here, we defined the binding and cleavage mechanism for Pro-Gly-Pro at the active site of LTA4H. Moreover, we designed a small molecule that selectively blocks synthesis of LTB 4 , although sparing the peptidase activity for inactivation of Pro-Gly-Pro, thus representing a novel type of LTA4H inhibitor that may pave the way for development of better treatments of inflammatory diseases.
The conformational flexibility and dynamics of two (1-->6)-linked disaccharides that are related to the action of the glycosyl transferase GnT-V have been investigated. NMR NOE and T-ROE spectroscopy experiments, conformation-dependent coupling constants and molecular dynamics (MD) simulations were used in the analyses. To facilitate these studies, the compounds were synthesised as alpha-d-[6-(13)C]-Manp-OMe derivatives, which reduced the (1)H NMR spectral overlap and facilitated the determination of two- and three-bond (1)H,(1)H, (1)H,(13)C and (13)C,(13)C-coupling constants. The population distribution for the glycosidic omega torsion angle in alpha-d-Manp-(1-->6)-alpha-d-Manp-OMe for gt/gg/tg was equal to 45:50:5, whereas in alpha-d-Manp-OMe it was determined to be 56:36:8. The dynamic model that was generated for beta-d-GlcpNAc-(1-->6)-alpha-d-Manp-OMe by MD simulations employing the PARM22/SU01 CHARMM-based force field was in very good agreement with experimental observations. beta-d-GlcpNAc-(1-->6)-alpha-d-Manp-OMe is described by an equilibrium of populated states in which the phi torsion angle has the exo-anomeric conformation, the psi torsion angle an extended antiperiplanar conformation and the omega torsion angle a distribution of populations predominantly between the gauche-trans and the gauche-gauche conformational states (i.e., gt/gg/tg) is equal to 60:35:5, respectively. The use of site-specific (13)C labelling in these disaccharides leads to increased spectral dispersion, thereby making NMR spectroscopy based conformational analysis possible that otherwise might be difficult to attain.
Feline hip dysplasia (FHD) is a debilitating condition affecting the hip joints of millions of domestic cats worldwide. Despite this, little is known about FHD except that it is relatively common in the large breed Maine Coon. We used 20 years of data from 5038 pedigree-registered Maine Coon cats in a radiographic health screening programme for FHD to determine, for the first time, its heritability, genetic correlation to body mass and response to selection. FHD prevalence was 37.4%, with no sex predilection; however, FHD severity increased with age and body mass. Heritability of the radiographic categories used to classify FHD severity was 0.36 (95%CI: 0.30–0.43). The severity of FHD symptoms was also genetically correlated with body mass (0.285), suggesting that selection for a large body type in this breed concurrently selects for FHD. Support for this was found by following generational responses to selective breeding against FHD. Not only did selective breeding successfully reduce the severity of FHD symptoms in descendants, but these cats were also smaller than their ancestors (−33g per generation). This study highlights the value of breeding programmes against FHD and cautions against breed standards that actively encourage large bodied cats.
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