Phosphate plays a crucial role in biology because of the stability of the phosphate ester bond. To overcome this inherent stability, enzymes that catalyze phosphoryl transfer reactions achieve ...enormous rate accelerations to operate on biologically relevant time scales, and the mechanisms that underpin catalysis have been the subject of extensive debate. In an archetypal system, β‑phosphoglucomutase catalyzes the reversible isomerization of β‑glucose 1-phosphate and glucose 6-phosphate via two phosphoryl transfer steps using a β‑glucose 1,6-bisphosphate intermediate and a catalytic MgII ion. In the present work, a variant of β-phosphoglucomutase, where the aspartate residue that acts as a general acid–base is replaced with asparagine, traps highly stable complexes containing the β-glucose 1,6-bisphosphate intermediate in the active site. Crystal structures of these complexes show that, when the enzyme is unable to transfer a proton, the intermediate is arrested in catalysis at an initial stage of phosphoryl transfer. The nucleophilic oxygen and transferring phosphorus atoms are aligned and in van der Waals contact, yet the enzyme is less closed than in transition-state (analogue) complexes, and binding of the catalytic MgII ion is compromised. Together, these observations indicate that optimal closure and optimal MgII binding occur only at higher energy positions on the reaction trajectory, allowing the enzyme to balance efficient catalysis with product dissociation. It is also confirmed that the general acid–base ensures that mutase activity is ∼103 fold greater than phosphatase activity in β-phosphoglucomutase.
We present the nuclear Overhauser effect-based structure determination of the Q41N variant of ubiquitin at 2500 bar, where the alternatively folded N2 state is 97% populated. This allows us to ...characterize the structure of the “pure” N2 state of ubiquitin. The N2 state shows a substantial change in the orientation of strand β5 compared to that of the normal folded N1 state, which matches the changes seen upon binding of ubiquitin to ubiquitin-activating enzyme E1. The recognition of E1 by ubiquitin is therefore best explained by conformational selection rather than induced-fit motion.
Abstract
Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate ...selectivity involves passage of the 5′-terminus/flap through the arch and recognition of a single nucleotide 3′-flap by the α2-α3 loop. Using NMR spectroscopy, we show that the solution conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2-α3 loop are disordered without substrate. Disorder within the arch explains how 5′-flaps can pass under it. NMR and single-molecule FRET data show a shift in the conformational ensemble in the arch and loop region upon addition of DNA. Furthermore, the addition of divalent metal ions to the active site of the hFEN1-DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1-DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.
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 AlF x -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 β-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.
Polyphenols (tannins) in the diet not only precipitate oral proteins, producing an astringent sensation, but also interact with dietary proteins and digestive enzymes in the gut, resulting in a ...variety of antinutritive and toxic effects. Salivary proline-rich proteins (PRPs), which are secreted into the oral cavity, form complexes with and precipitate dietary polyphenols, and thus, they constitute the primary mammalian defense directed against ingested tannins. In order to characterize the interaction, NMR studies were performed which involved titrating a series of polyphenols into a synthetic 19-residue PRP fragment. The results show that the predominant mode of association is a hydrophobic stacking of the polyphenol ring against the pro-S face of proline and that the first proline residue of a Pro-Pro sequence is a particularly favored binding site. Measurement of dissociation constants indicates that the larger and more complex polyphenols interact more strongly with the PRP fragment; the order of binding affinity was determined as procyanidin dimer B-2 > pentagalloylglucose > trigalloylglucose >> proanthocyanidin monomer (−)-epicatechin ≈ propyl gallate. Smaller polyphenols can bind with one phenolic ring stacked against each proline residue, whereas larger polyphenols occupy two or three consecutive prolines. The more complex polyphenols interact with the PRP fragment in a multidentate fashion; moreover, they self-associate or stack when bound. Thus, a model is proposed in which multiple polyphenol/polyphenol and polyphenol/PRP interactions act cooperatively to achieve precipitation.
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 (MgF3 −) and tetrafluoroaluminate (AlF4 −) complexes of PGK have been determined using X-ray crystallography and 19F-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 α-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 (AlF3 0) complex. The AlF3 0 moiety retains the octahedral geometry observed within AlF4 − TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF3 0 complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF3 −.
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.
Enterococcus faecalis
is a major causative agent of hospital acquired infections. The ability of
E. faecalis
to evade the host immune system is essential during pathogenesis, which has been shown to ...be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of
E. faecalis
, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in
E. faecalis
virulence.
We have used NMR and computational methods to characterize the dynamics of the ribonuclease barnase over a wide range of timescales in free and inhibitor-bound states. Using temperature- and ...denaturant-dependent measurements of chemical shift, we show that barnase undergoes frequent and highly populated hinge bending. Using relaxation dispersion, we characterize a slower and less populated motion with a rate of 750 ± 200 s
, involving residues around the lip of the active site, which occurs in both free and bound states and therefore suggests conformational selection. Normal mode calculations characterize correlated hinge bending motions on a very rapid timescale. These three measurements are combined with previous measurements and molecular dynamics calculations on barnase to characterize its dynamic landscape on timescales from picoseconds to milliseconds and length scales from 0.1 to 2.5 nm. We show that barnase has two different large-scale fluctuations: one on a timescale of 10
-10
s that has no free energy barrier and is a hinge bending that is determined by the architecture of the protein; and one on a timescale of milliseconds (i.e., 750 s
) that has a significant free energy barrier and starts from a partially hinge-bent conformation. These two motions can be described as hierarchical, in that the more highly populated faster motion provides a platform for the slower (less probable) motion. The implications are discussed. The use of temperature and denaturant is suggested as a simple and general way to characterize motions on the intermediate ns-μs timescale.
The effects of sodium thiocyanate, sodium chloride, and sodium sulfate on the ribonuclease barnase were studied using differential scanning calorimetry (DSC) and NMR. Both measurements reveal ...specific and saturable binding at low anion concentrations (up to 250 mM), which produces localized conformational and energetic effects that are unrelated to the Hofmeister series. The binding of sulfate slows intramolecular motions, as revealed by peak broadening in 13C heteronuclear single quantum coherence spectroscopy. None of the anions shows significant binding to hydrophobic groups. Above 250 mM, the DSC results are consistent with the expected Hofmeister effects in that the chaotropic anion thiocyanate destabilizes barnase. In this higher concentration range, the anions have approximately linear effects on protein NMR chemical shifts, with no evidence for direct interaction of the anions with the protein surface. We conclude that the effects of the anions on barnase are mediated by solvent interactions. The results are not consistent with the predictions of the preferential interaction, preferential hydration, and excluded volume models commonly used to describe Hofmeister effects. Instead, they suggest that the Hofmeister anion effects on both stability and solubility of barnase are due to the way in which the protein interacts with water molecules, and in particular with water dipoles, which are more ordered around sulfate anions and less ordered around thiocyanate anions.
PDF
