Nicola J. Baxter

Temperature dependence of 1H chemical shifts in proteins

Temperature coefficients have been measured by 2D NMR methods forthe amide and CαH proton chemical shifts in two globularproteins, bovine pancreatic trypsin inhibitor and hen egg-white lysozyme.The temperature-dependent changes in chemical shift are generally linear upto about 15° below the global denaturation temperature, and the derivedcoefficients span a range of roughly −16 to +2 ppb/K for amide protonsand −4 to +3 ppb/K for CαH. The temperaturecoefficients can be rationalized by the assumption that heating causesincreases in thermal motion in the protein. Precise calculations oftemperature coefficients derived from protein coordinates are not possible,since chemical shifts are sensitive to small changes in atomic coordinates.Amide temperature coefficients correlate well with the location of hydrogenbonds as determined by crystallography. It is concluded that a combined useof both temperature coefficients and exchange rates produces a far morereliable indicator of hydrogen bonding than either alone. If an amide protonexchanges slowly and has a temperature coefficient more positive than−4.5 ppb/K, it is hydrogen bonded, while if it exchanges rapidly andhas a temperature coefficient more negative than −4.5 ppb/K, it is nothydrogen bonded. The previously observed unreliability of temperaturecoefficients as measures of hydrogen bonding in peptides may arise fromlosses of peptide secondary structure on heating.

Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones

The translationally controlled tumor-associated proteins (TCTPs) are a highly conserved and abundantly expressed family of eukaryotic proteins that are implicated in both cell growth and the human acute allergic response but whose intracellular biochemical function has remained elusive. We report here the solution structure of the TCTP from Schizosaccharomyces pombe, which, on the basis of sequence homology, defines the fold of the entire family. We show that TCTPs form a structural superfamily with the Mss4/Dss4 family of proteins, which bind to the GDP/GTP free form of Rab proteins (members of the Ras superfamily) and have been termed guanine nucleotide-free chaperones (GFCs). Mss4 also acts as a relatively inefficient guanine nucleotide exchange factor (GEF). We further show that the Rab protein binding site on Mss4 coincides with the region of highest sequence conservation in the TCTP family. This is the first link to any other family of proteins that has been established for the TCTP family and suggests the presence of a GFC/GEF at extremely high abundance in eukaryotic cells.

Tannin interactions with a full‐length human salivary proline‐rich protein display a stronger affinity than with single proline‐rich repeats

The protein IB5 has been purified from human parotid saliva. This protein contains several repeats of a short proline‐rich sequence. Dissociation constants have been measured at several discrete binding sites using 1H‐NMR for the hydrolysable tannins (polyphenols) ( , and and the condensed proanthocyanidin (−)‐epicatechin. The dissociation constants for trigalloyl glucose and pentagalloyl glucose were 15 × 10−5 and 1.7 × 10−5 M, respectively, which are 115 and 1660 times stronger than those previously measured under the same conditions for a single repeat of a mouse salivary proline‐rich protein. The increase in affinity is ascribed to intramolecular secondary interactions, which are strengthened by the rigidity of the interacting molecules.

Transition State Analogue Structures of Human Phosphoglycerate Kinase Establish the Importance of Charge Balance in Catalysis.

Transition state analogue (TSA) complexes formed by phosphoglycerate kinase (PGK) have been used to test the hypothesis that balancing of charge within the transition state dominates enzyme-catalyzed phosphoryl transfer. High-resolution structures of trifluoromagnesate (MgF(3)(-)) and tetrafluoroaluminate (AlF(4)(-)) complexes of PGK have been determined using X-ray crystallography and (19)F-based NMR methods, revealing the nature of the catalytically relevant state of this archetypal metabolic kinase. Importantly, the side chain of K219, which coordinates the alpha-phosphate group in previous ground state structures, is sequestered into coordinating the metal fluoride, thereby creating a charge environment complementary to the transferring phosphoryl group. In line with the dominance of charge balance in transition state organization, the substitution K219A induces a corresponding reduction in charge in the bound aluminum fluoride species, which changes to a trifluoroaluminate (AlF(3)(0)) complex. The AlF(3)(0) moiety retains the octahedral geometry observed within AlF(4)(-) TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF(3)(0) complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF(3)(-).

Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF-3 rather than by phosphoranes.

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of β-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO 3 - , but is MgF 3 - (trifluoromagnesate). Although MgF 3 - complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using F 19 NMR. Measurements on three β - PGM - MgF 3 - -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F ⋯ H - N scalar couplings, and the atomic positions determined from the high-resolution crystal structure of the β - PGM - MgF 3 - - G 6 P complex. The measurements provide independent validation of the structural and isoelectronic MgF 3 - model of near-transition state conformations.

Stacking interactions between caffeine and methyl gallate

The self-association of caffeine and methyl gallate (a simple polyphenol) has been studied by NMR in the solvent mixture H2O–[2H6]-dimethylsulfoxide (9 : 1, by volume) at 288 K, using the isodesmic model (also known as the indefinite non-cooperative model). The chemical shift changes observed suggest that caffeine molecules in a stack are associated in a head-to-tail (antiparallel) manner. The interaction between monomeric caffeine and methyl gallate stacks has also been investigated and the results indicate that caffeine prefers to bind at the ends of methyl gallate stacks rather than intercalating into the stacks. Comparison of the association constants suggests that in a mixture of methyl gallate and caffeine, heterostacks are formed in preference to self-associated solutes, which has an obvious bearing on the bioavailability of aromatic and phenolic species.

Molecular basis for bacterial peptidoglycan recognition by LysM domains.

Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM–peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms.

Anionic charge is prioritized over geometry in aluminum and magnesium fluoride transition state analogs of phosphoryl transfer enzymes.

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.

A Trojan horse transition state analogue generated by MgF3− formation in an enzyme active site

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. β-Phosphoglucomutase catalyses the isomerization of β-glucose-1-phosphate to β-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using 19F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF3− mimics the transferring PO3− moiety. Here we present a detailed characterization of the metal ion–fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of β-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.

Structure and dynamics of coxsackievirus B4 2A proteinase, an enyzme involved in the etiology of heart disease.

ABSTRACT The 2A proteinases (2Apro) from the picornavirus family are multifunctional cysteine proteinases that perform essential roles during viral replication, involving viral polyprotein self-processing and shutting down host cell protein synthesis through cleavage of the eukaryotic initiation factor 4G (eIF4G) proteins. Coxsackievirus B4 (CVB4) 2Apro also cleaves heart muscle dystrophin, leading to cytoskeletal dysfunction and the symptoms of human acquired dilated cardiomyopathy. We have determined the solution structure of CVB4 2Apro (extending in an N-terminal direction to include the C-terminal eight residues of CVB4 VP1, which completes the VP1-2Apro substrate region). In terms of overall fold, it is similar to the crystal structure of the mature human rhinovirus serotype 2 (HRV2) 2Apro, but the relatively low level (40%) of sequence identity leads to a substantially different surface. We show that differences in the cI-to-eI2 loop between HRV2 and CVB4 2Apro translate to differences in the mechanism of eIF4GI recognition. Additionally, the nuclear magnetic resonance relaxation properties of CVB4 2Apro, particularly of residues G1 to S7, F64 to S67, and P107 to G111, reveal that the substrate region is exchanging in and out of a conformation in which it occupies the active site with association and dissociation rates in the range of 100 to 1,000 s−1. This exchange influences the conformation of the active site and points to a mechanism for how self-processing can occur efficiently while product inhibition is avoided.