Publications
Below is a selection of recent representative publications from the research group that illustrate the scope and impact of our research.
Hay S, Sutcliffe MJ, Scrutton NS. (2007) Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects. PNAS 104, 507-12
Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar = 100 kPa) to 2 kbar accelerates the hydride transfer reaction when both protium (from 50 to 161 s(-1) at 25 degrees C) and deuterium (12 to 31 s(-1) at 25 degrees C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25 degrees C), an observation incompatible with the Bell correction model for hydrogen tunneling but consistent with a full tunneling model. By numerical modeling, we show that both the pressure and temperature dependencies of the reaction rates are consistent with the framework of the environmentally coupled tunneling model of Kuznetsov and Ulstrup [Kuznetsov AM, Ulstrup J (1999) Can J Chem 77:1085-1096], providing additional support for the role of a promoting motion in the hydride tunneling reaction in morphinone reductase. Our study demonstrates the utility of "barrier engineering" by using hydrostatic pressure as a probe for tunneling regimes in enzyme systems and provides added and independent support for the requirement of promoting motions in such tunneling reactions.
www: http://www.ncbi.nlm.nih.gov/pubmed/17202258?
Masgrau L, Roujeinikova A, Johannissen LO, Hothi P, Basran J, Ranaghan KE, Mulholland AJ, Sutcliffe MJ, Scrutton NS, Leys D. (2006) Atomic description of an enzyme reaction dominated by proton tunneling. Science 312, 237-241.
We present an atomic-level description of the reaction chemistry of an enzyme-catalyzed reaction dominated by proton tunneling. By solving structures of reaction intermediates at near-atomic resolution, we have identified the reaction pathway for tryptamine oxidation by aromatic amine dehydrogenase. Combining experiment and computer simulation, we show proton transfer occurs predominantly to oxygen O2 of Asp(128)beta in a reaction dominated by tunneling over approximately 0.6 angstroms. The role of long-range coupled motions in promoting tunneling is controversial. We show that, in this enzyme system, tunneling is promoted by a short-range motion modulating proton-acceptor distance and no long-range coupled motion is required.
Girvan HM, Heyes DJ, Scrutton NS, Munro AW. (2007) Laser photoexcitation of NAD(P)H induces reduction of P450 BM3 heme domain on the microsecond time scale. J. Am. Chem. Soc. 129, 6647-53
We demonstrate that photoexcitation of NAD(P)H at 355 nm using a Nd:YAG laser leads to rapid reduction of the heme domain of the Bacillus megaterium fatty acid hydroxylase flavocytochrome P450 BM3. An aqueous electron derived from photoexcited NAD(P)H is rapidly transferred to the heme domain, enabling the formation of a carbon monoxy complex of the ferrous P450 (FeII-CO) on the microsecond time scale. Using this approach we have determined the limiting rate constant (1770 s-1 for substrate-free heme domain) for formation of the FeII-CO complex. We find no dependence of the observed rate of FeII-CO complex formation on NAD(P)H concentration but demonstrate a hyperbolic dependence on carbon monoxide concentration. The apparent dissociation constant for the complex of carbon monoxide bound noncovalently to the ferric form of the BM3 heme domain (and with NADH as reductant) is 323 microM. Binding of a P450 substrate (N-palmitoylglycine) weakened the complex between carbon monoxide and the ferric BM3 heme domain (Kd increased to 1404 microM) but enhanced the rate of formation of the FeII-CO complex (3036 s-1 for substrate-free heme domain). This study demonstrates the applicability of NAD(P)H photoexcitation as a method for rapid electron delivery to P450 enzymes and provides a new route to probing the P450 catalytic cycle and its transient intermediates.
Gilbert RJ, Gordiyenko Y, von der Haar T, Sonnen AF, Hofmann G, Nardelli M, Stuart DI, McCarthy JE. (2007) Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex. PNAS 104, 5788-93
In the process of protein synthesis, the small (40S) subunit of the eukaryotic ribosome is recruited to the capped 5' end of the mRNA, from which point it scans along the 5' untranslated region in search of a start codon. However, the 40S subunit alone is not capable of functional association with cellular mRNA species; it has to be prepared for the recruitment and scanning steps by interactions with a group of eukaryotic initiation factors (eIFs). In budding yeast, an important subset of these factors (1, 2, 3, and 5) can form a multifactor complex (MFC). Here, we describe cryo-EM reconstructions of the 40S subunit, of the MFC, and of 40S complexes with MFC factors plus eIF1A. These studies reveal the positioning of the core MFC on the 40S subunit, and show how eIF-binding induces mobility in the head and platform and reconfigures the head-platform-body relationship. This is expected to increase the accessibility of the mRNA channel, thus enabling the 40S subunit to convert to a recruitment-competent state.
Dittrich S, Mitchell SL, Blagborough AM, Wang Q, Wang P, Sims PF, Hyde JE. (2008) An atypical orthologue of 6-pyruvoyltetrahydropterin synthase can provide the missing link in the folate biosynthesis pathway of malaria parasites. Mol Microbiol. 67, 609-618
Folate metabolism in malaria parasites is a long-standing, clinical target for chemotherapy and prophylaxis. However, despite determination of the complete genome sequence of the lethal species Plasmodium falciparum, the pathway of de novo folate biosynthesis remains incomplete, as no candidate gene for dihydroneopterin aldolase (DHNA) could be identified. This enzyme catalyses the third step in the well-characterized pathway of plants, bacteria, and those eukaryotic microorganisms capable of synthesizing their own folate. Utilizing bioinformatics searches based on both primary and higher protein structures, together with biochemical assays, we demonstrate that P. falciparum cell extracts lack detectable DHNA activity, but that the parasite possesses an unusual orthologue of 6-pyruvoyltetrahydropterin synthase (PTPS), which simultaneously gives rise to two products in comparable amounts, the predominant of which is 6-hydroxymethyl-7,8-dihydropterin, the substrate for the fourth step in folate biosynthesis (catalysed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; PPPK). This can provide a bypass for the missing DHNA activity and thus a means of completing the biosynthetic pathway from GTP to dihydrofolate. Supported by site-directed mutagenesis experiments, we ascribe the novel catalytic activity of the malarial PTPS to a Cys to Glu change at its active site relative to all previously characterized PTPS molecules, including that of the human host.
Blundell CD, Mahoney DJ, Cordell MR, Almond A, Kahmann JD, Perczel A, Taylor JD, Campbell ID, Day AJ. (2007) Determining the molecular basis for the pH-dependent interaction between the link module of human TSG-6 and hyaluronan. J. Biol. Chem. 282, 12976-12988
TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His(4)) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pK(a) values of charged residues within the Link module were determined using NMR. These data indicated that His(4) makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp(89)), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr(12)). This molecular network transmits the change in ionization state of His(4) to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His(45), is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments.
Jennings MD, Blankley RT, Baron M, Golovanov AP, Avis JM. (2007) Specificity and autoregulation of Notch binding by tandem WW domains in suppressor of Deltex. J. Biol. Chem. 282, 29032-29042
WW domains target proline-tyrosine (PY) motifs and frequently function as tandem pairs. When studied in isolation, single WW domains are notably promiscuous and regulatory mechanisms are undoubtedly required to ensure selective interactions. Here, we show that the fourth WW domain (WW4) of Suppressor of Deltex, a modular Nedd4-like protein that down-regulates the Notch receptor, is the primary mediator of a direct interaction with a Notch-PY motif. A natural Trp to Phe substitution in WW4 reduces its affinity for general PY sequences and enhances selective interaction with the Notch-PY motif via compensatory specificity-determining interactions with PY-flanking residues. When WW4 is paired with WW3, domain-domain association, impeding proper folding, competes with Notch-PY binding to WW4. This novel mode of autoinhibition is relieved by binding of another ligand to WW3. Such cooperativity may facilitate the transient regulatory interactions observed in vivo between Su(dx) and Notch in the endocytic pathway. The highly conserved tandem arrangement of WW domains in Nedd4 proteins, and similar arrangements in more diverse proteins, suggests domain-domain communication may be integral to regulation of their associated cellular activities.
Bray T, Doig AJ, Warwicker J. (2008) Sequence and structural features of enzymes and their active sites by EC class. J. Mol. Biol. 386, 1423-36
We have analysed a non-redundant set of 294 enzymes for differences in sequence and structural features between the six main Enzyme Commission (EC) classification groups. This systematic study of enzymes, and their active sites in particular, aims to increase understanding of how the structure of an enzyme relates to its functional role. Many features showed significant differences between the EC classes, including active-site polarity, enzyme size and active-site amino acid propensities. Many attributes correlate with each other to form clusters of related features from which we chose representative features for further analysis. Oxidoreductases have more non-polar active sites, which can be attributed to cofactor binding and a preference for Glu over Asp in active sites in comparison to the other classes. Lyases form a significantly higher proportion of oligomers than any other class, whilst the hydrolases form the largest proportion of monomers. These features were then used in a prediction model that classified each enzyme into its top EC class with an accuracy of 33.1%, which is an increase of 16.4% over random classification. Understanding the link between structure and function is critical to improving enzyme design and the prediction of protein function from structure without transfer of annotation from alignments.
Stepien P, Johnson GN. (2009) Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte thellungiella: role of the plastid terminal oxidase as an alternative electron sink. Plant Physiol. 149, 1154-1165
The effects of short-term salt stress on gas exchange and the regulation of photosynthetic electron transport were examined in Arabidopsis (Arabidopsis thaliana) and its salt-tolerant close relative Thellungiella (Thellungiella halophila). Plants cultivated on soil were challenged for 2 weeks with NaCl. Arabidopsis showed a much higher sensitivity to salt than Thellungiella; while Arabidopsis plants were unable to survive exposure to greater than 150 mM salt, Thellugiella could tolerate concentrations as high as 500 mM with only minimal effects on gas exchange. Exposure of Arabidopsis to sublethal salt concentrations resulted in stomatal closure and inhibition of CO2 fixation. This lead to an inhibition of electron transport though photosystem II (PSII), an increase in cyclic electron flow involving only PSI, and increased nonphotochemical quenching of chlorophyll fluorescence. In contrast, in Thellungiella, although gas exchange was marginally inhibited by high salt and PSI was unaffected, there was a large increase in electron flow involving PSII. This additional electron transport activity is oxygen dependent and sensitive to the alternative oxidase inhibitor n-propyl gallate. PSII electron transport in Thellungiella showed a reduced sensitivity to 2'-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenylether, an inhibitor of the cytochrome b6f complex. At the same time, we observed a substantial up-regulation of a protein reacting with antibodies raised against the plastid terminal oxidase. No such up-regulation was seen in Arabidopsis. We conclude that in salt-stressed Thellungiella, plastid terminal oxidase acts as an alternative electron sink, accounting for up to 30% of total PSII electron flow.
Baxter NJ, Blackburn GM, Marston JP, Hounslow AM, Cliff MJ, Bermel W, Williams NH, Hollfelder F, Wemmer DE, Waltho JP. (2008) Anionic charge is prioritized over geometry in aluminum and magnesium fluoride transition state analogs of phosphoryl transfer enzymes. J. Am. Chem. Soc. 130, 3952-3958
Phosphoryl transfer reactions are ubiquitous in biology and metal fluoride complexes have played a central role in structural approaches to understanding how they are catalyzed. In particular, numerous structures of AlFx-containing complexes have been reported to be transition state analogs (TSAs). A survey of nucleotide kinases has proposed a correlation between the pH of the crystallization solution and the number of coordinated fluorides in the resulting aluminum fluoride TSA complexes formed. Enzyme ligands crystallized above pH 7.0 were attributed to AlF3, whereas those crystallized at or below pH 7.0 were assigned as AlF4-. We use 19F NMR to show that for beta-phosphoglucomutase from Lactococcus lactis, the pH-switch in fluoride coordination does not derive from an AlF4- moiety converting into AlF3. Instead, AlF4- is progressively replaced by MgF3- as the pH increases. Hence, the enzyme prioritizes anionic charge at the expense of preferred native trigonal geometry over a very broad range of pH. We demonstrate similar behavior for two phosphate transfer enzymes that represent typical biological phosphate transfer catalysts: an amino acid phosphatase, phosphoserine phosphatase from Methanococcus jannaschii and a nucleotide kinase, phosphoglycerate kinase from Geobacillus stearothermophilus. Finally, we establish that at near-physiological ratios of aluminum to magnesium, aluminum can dominate over magnesium in the enzyme-metal fluoride inhibitory TSA complexes, and hence is the more likely origin of some of the physiological effects of fluoride.
Berry R, Jowitt TA, Ferrand J, Roessle M, Grossmann JG, Canty-Laird EG, Kammerer RA, Kadler KE, Baldock C. (2009) Role of dimerization and substrate exclusion in the regulation of bone morphogenetic protein-1 and mammalian tolloid. PNAS 106, 8561-8566
The bone morphogenetic protein (BMP)-1/tolloid metalloproteinases are evolutionarily conserved enzymes that are fundamental to dorsal-ventral patterning and tissue morphogenesis. The lack of knowledge regarding how these proteinases recognize and cleave their substrates represents a major hurdle to understanding tissue assembly and embryonic patterning. Although BMP-1 and mammalian tolloid (mTLD) are splice variants, it is puzzling why BMP-1, which lacks 3 of the 7 noncatalytic domains present in all other family members, is the most effective proteinase. Using a combination of single-particle electron microscopy, small-angle X-ray scattering, and other biophysical measurements in solution, we show that mTLD, but not BMP-1, forms a calcium-ion-dependent dimer under physiological conditions. Using a domain deletion approach, we provide evidence that EGF2, which is absent in BMP-1, is critical to the formation of the dimer. Based on a combination of structural and functional data, we propose that mTLD activity is regulated by a substrate exclusion mechanism. These results provide a mechanistic insight into how alternative splicing of the Bmp1 gene produces 2 proteinases with differing biological activities and have broad implications for regulation of BMP-1/mTLD and related proteinases during BMP signaling and tissue assembly.
Williams SG, Lovell SC. (2009) The effect of sequence evolution on protein structural divergence. Mol. Biol. Evol. 26, 1055-1065
The complex constraints imposed by protein structure and function result in varied rates of sequence and structural divergence in proteins. Analysis of sequence differences between homologous proteins can advance our understanding of structural divergence and some of the constraints that govern the evolution of these molecules. Here, we assess the relationship between amino acid sequence and structural divergence. Firstly, we demonstrate that the relationship between protein sequence and structural divergence is governed by a variety of evolutionary constraints, including solvent exposure and secondary structure. Secondly, although compensatory substitutions are widespread, we find many radical size-changing mutations that are not compensated by neighboring complementary changes. Instead, these noncompensated substitutions are mitigated by alteration of protein structure. These results suggest a combined mechanism of accommodating substitutions in proteins, involving both coevolution and structural accommodation. Such a mechanism can explain previously observed correlated substitutions of residues that are distant both in sequence and structure, allowing an integrated view of sequence and structural divergence of proteins.
Laing E, Sidhu K, Hubbard SJ. (2008) Predicted transcription factor binding sites as predictors of operons in Escherichia coli and Streptomyces coelicolor. BMC Genomics 12, 79.
BACKGROUND: As a polycistronic transcriptional unit of one or more adjacent genes, operons play a key role in regulation and function in prokaryotic biology, and a better understanding of how they are constituted and controlled is needed. Recent efforts have attempted to predict operonic status in sequenced genomes using a variety of techniques and data sources. To date, non-homology based operon prediction strategies have mainly used predicted promoters and terminators present at the extremities of transcriptional unit as predictors, with reasonable success. However, transcription factor binding sites (TFBSs), typically found upstream of the first gene in an operon, have not yet been evaluated. RESULTS: Here we apply a method originally developed for the prediction of TFBSs in Escherichia coli that minimises the need for prior knowledge and tests its ability to predict operons in E. coli and the 'more complex', pharmaceutically important, Streptomyces coelicolor. We demonstrate that through building genome specific TFBS position-specific-weight-matrices (PSWMs) it is possible to predict operons in E. coli and S. coelicolor with 83% and 93% accuracy respectively, using only TFBS as delimiters of operons. Additionally, the 'palindromicity' of TFBS footprint data of E. coli is characterised. CONCLUSION: TFBS are proposed as novel independent features for use in prokaryotic operon prediction (whether alone or as part of a set of features) given their efficacy as operon predictors in E. coli and S. coelicolor. We also show that TFBS footprint data in E. coli generally contains inverted repeats with significantly (p < 0.05) greater palindromicity than random sequences. Consequently, the palindromicity of putative TFBSs predicted can also enhance operon predictions.