Matthew W. Bowler

Ground State Structure of F1-ATPase from Bovine Heart Mitochondria at 1.9 A Resolution

The structure of bovine F1-ATPase, crystallized in the presence of AMP-PNP and ADP, but in the absence of azide, has been determined at 1.9Å resolution. This structure has been compared with the previously described structure of bovine F1-ATPase determined at 1.95Å resolution with crystals grown under the same conditions but in the presence of azide. The two structures are extremely similar, but they differ in the nucleotides that are bound to the catalytic site in the βDP-subunit. In the present structure, the nucleotide binding sites in the βDP- and βTP-subunits are both occupied by AMP-PNP, whereas in the earlier structure, the βTP site was occupied by AMP-PNP and the βDP site by ADP, where its binding is enhanced by a bound azide ion. Also, the conformation of the side chain of the catalytically important residue, αArg-373 differs in the βDP- and βTP-subunits. Thus, the structure with bound azide represents the ADP inhibited state of the enzyme, and the new structure represents a ground state intermediate in the active catalytic cycle of ATP hydrolysis.

MxCuBE: a synchrotron beamline control environment customized for macromolecular crystallography experiments

MxCuBE is a beamline control environment optimized for the needs of macromolecular crystallography. This paper describes the design of the software and the features that MxCuBE currently provides.

Improving diffraction by humidity control: a novel device compatible with X-ray beamlines

Dehydration of protein crystals is rarely used, despite being a post-crystallization method that is useful for the improvement of crystal diffraction properties, as it is difficult to reproduce and monitor. A novel device for hydration control of macromolecular crystals in a standard data-collection environment has been developed. The device delivers an air stream of precise relative humidity that can be used to alter the amount of water in macromolecular crystals. The device can be rapidly installed and is fully compatible with most standard synchrotron X-ray beamlines. Samples are mounted in cryoloops and the progress of dehydration can be monitored both optically and by the acquisition of diffraction images. Once the optimal hydration level has been obtained, cryocooling is easy to achieve by hand or by using a sample changer. The device has been thoroughly tested on several ESRF beamlines and is available to users.

Diffraction cartography: applying microbeams to macromolecular crystallography sample evaluation and data collection

Crystals of biological macromolecules often exhibit considerable inter-crystal and intra-crystal variation in diffraction quality. This requires the evaluation of many samples prior to data collection, a practice that is already widespread in macromolecular crystallography. As structural biologists move towards tackling ever more ambitious projects, new automated methods of sample evaluation will become crucial to the success of many projects, as will the availability of synchrotron-based facilities optimized for high-throughput evaluation of the diffraction characteristics of samples. Here, two examples of the types of advanced sample evaluation that will be required are presented: searching within a sample-containing loop for microcrystals using an X-ray beam of 5 microm diameter and selecting the most ordered regions of relatively large crystals using X-ray beams of 5-50 microm in diameter. A graphical user interface developed to assist with these screening methods is also presented. For the case in which the diffraction quality of a relatively large crystal is probed using a microbeam, the usefulness and implications of mapping diffraction-quality heterogeneity (diffraction cartography) are discussed. The implementation of these techniques in the context of planned upgrades to the ESRFs structural biology beamlines is also presented.

Why did Nature select phosphate for its dominant roles in biology

Evolution has placed phosphate mono- and diesters at the heart of biology. The enormous diversity of their roles has called for the evolution of enzyme catalysts for phosphoryl transfer that are among the most proficient known. A combination of high-resolution X-ray structure analysis and 19F NMR definition of metal fluoride complexes of such enzymes, that are mimics of the transition state for the reactions catalysed, has delivered atomic detail of the nature of such catalysis for a range of phosphoryl transfer processes. The catalytic simplicity thus revealed largely explains the paradox of the contrast between the extreme stability of structural phosphate esters and the lability of phosphates in regulation and signalling processes. A brief survey of the properties of oxyacids and their esters for other candidate elements for these vital roles fails to identify a suitable alternative to phosphorus, thereby underpinning Todd’s Hypothesis “Where there’s life there’s phosphorus” as a statement of truly universal validity.

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.

Automatic processing of macromolecular crystallography X-ray diffraction data at the ESRF

A system for the automatic reduction of single- and multi-position macromolecular crystallography data is presented.

Inducing phase changes in crystals of macromolecules: Status and perspectives for controlled crystal dehydration

The increase in the number of large multi-component complexes and membrane protein crystal structures determined over the last few years can be ascribed to a number of factors such as better protein expression and purification systems, the emergence of high-throughput crystallization techniques and the advent of 3rd generation synchrotron sources. However, many systems tend to produce crystals that can be extremely heterogeneous in their diffraction properties. This prevents, in many cases, the collection of diffraction data of sufficient quality to yield useful biological or phase information. Techniques that can increase the diffraction quality of macromolecular crystals can therefore be essential in the successful conclusion of these challenging projects. No technique is universal but encouraging results have been recently achieved by carrying out the controlled dehydration of crystals of biological macromolecules. A new device that delivers a stream of air with a precisely controlled relative humidity to the complicated sample environment found at modern synchrotron beamlines has been conceived at the EMBL Grenoble and developed by the EMBL and the ESRF as part of the SPINE2 complexes project, a European Commission funded protein structure initiative. The device, the HC1b, has been available for three years at the ESRF macromolecular crystallography beamlines and many systems have benefitted from on-line controlled dehydration. Here we describe a standard dehydration experiment, highlight some successful cases and discuss the different possible uses of the device.

Structural basis of the PNRC2-mediated link between mrna surveillance and decapping.

Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance system, and human PNRC2 protein mediates the link between mRNA surveillance and decapping. However, the mechanism by which PNRC2 interacts with the mRNA surveillance machinery and stimulates NMD is unknown. Here, we present the crystal structure of Dcp1a in complex with PNRC2. The proline-rich region of PNRC2 is bound to the EVH1 domain of Dcp1a, while its NR-box mediates the interaction with the hyperphosphorylated Upf1. The mode of PNRC2 interaction with Dcp1a is distinct from those observed in other EVH1/proline-rich ligands interactions. Disruption of the interaction of PNRC2 with Dcp1a abolishes its P-body localization and ability to promote mRNA degradation when tethered to mRNAs. PNRC2 acts in synergy with Dcp1a to stimulate the decapping activity of Dcp2 by bridging the interaction between Dcp1a and Dcp2, suggesting that PNRC2 is a decapping coactivator in addition to its adaptor role in NMD.