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1.  High-resolution structures of Trypanosoma brucei pteridine reductase ligand complexes inform on the placement of new molecular entities in the active site of a potential drug target 
Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species. These protozoa cause serious diseases for which current therapies are inadequate. High-resolution structures have been determined, using data between 1.6 and 1.1 Å resolution, of T. brucei PTR1 in complex with pemetrexed, trimetrexate, cyromazine and a 2,4-diaminopyrimidine derivative. The structures provide insight into the interactions formed by new molecular entities in the enzyme active site with ligands that represent lead compounds for structure-based inhibitor development and to support early-stage drug discovery.
doi:10.1107/S0907444910040886
PMCID: PMC3655514  PMID: 21123874
2.  Extending the usability of the phasing power of diselenide bonds: SeCys SAD phasing of CsgC using a non-auxotrophic strain 
The CsgC protein is a component of the curli system in Escherichia coli. Reported here is the successful incorporation of selenocysteine (SeCys) and selenomethionine (SeMet) into recombinant CsgC, yielding derivatised crystals suitable for structural determination. Unlike in previous reports, a standard, autotrophic expression strain was used and only single anomalous diffraction (SAD) data were required for successful phasing. The level of SeCys/SeMet incorporation was estimated by mass spectrometry to be about 80%. Native protein crystallised in two different crystal forms (C2221, form 1 and C21, form 2) both diffracting to 2.4 Å, whilst Se-derivatised protein crystallised in C21 and diffracted to 1.7 Å. The Se-derivatised crystals are suitable for SAD structure determination using only anomalous signal derived from the selenocysteine residues. These results extend the usability of SeCys labelling to more general and less favourable cases, rendering it a suitable alternative to traditional phasing approaches.
doi:10.1107/S0907444910042022
PMCID: PMC3522112  PMID: 21206057
selenocysteine; SAD; non-auxotrophic E. coli strains; CsgC protein
3.  Structural analysis of Pneumocystis carinii and human DHFR complexes with NADPH and a series of five potent 6-[5′-(ω-carboxyalkoxy)benzyl]pyrido[2,3-d]pyrimidine derivatives 
These data reveal that the ethyl esters of a series of 5′-(ω-carboxyalkoxy)benzylpyrido[2,3-d]pyrimidines have flexible side-chain conformations that do not optimize interactions with Arg75 in pcDHFR. Also, a novel conformation not reported in other DHFR structures was observed for one conformer of one of the compounds that had a disordered side chain.
Structural data are reported for five antifolates, namely 2,4-diamino-6-[5′-(5-carboxypentyloxy)-2′-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine, (1), and the 5′-[3-(ethoxycarbonyl)propoxy]-, (2), 5′-[3-(ethoxycarbonyl)butoxy]-, (3), 5′-[3-(ethoxycarbonyl)pentyloxy]-, (4), and 5′-benzyloxy-, (5), derivatives, which are potent and selective for Pneumocystis carinii dihydrofolate reductase (pcDHFR). Crystal structures are reported for their ternary complexes with NADPH and pcDHFR refined to between 1.4 and 2.0 Å resolution and for that of 3 with human DHFR (hDHFR) to 1.8 Å resolution. These data reveal that the carboxylate of the ω-carboxyalkoxy side chain of 1, the most potent inhibitor in this series, forms ionic interactions with the conserved Arg75 in the substrate-binding pocket of pcDHFR, whereas the less potent ethyl esters of 2–4 bind with variable side-chain conformations. The benzyloxy side chain of 5 makes no contact with Arg75 and is the least active inhibitor in this series. These structural results suggest that the weaker binding of this series compared with that of their pyrimidine homologs in part arises from the flexibility observed in their side-chain conformations, which do not optimize intermolecular contact to Arg75. Structural data for the binding of 3 to both hDHFR and pcDHFR reveals that the inhibitor binds in two different conformations, one similar to each of the two conformations observed for the parent pyrido[2,3-d]pyrimidine, piritrexim (PTX), bound to hDHFR. The structure of the pcDHFR complex of 4 reveals disorder in the side-chain orientation; one orientation has the ω-carboxy­alkoxy side chain positioned in the folate-binding pocket similar to the others in this series, while the second orientation occupies a new site near the nicotinamide ring of NADPH. This alternate binding site has not been observed in other DHFR structures. Structural data for the pcDHFR complex of 5 show that its benzyl side chain forms intermolecular van der Waals interactions with Phe69 in the binding pocket that could account for its enhanced binding selectivity compared with the other analogs in this series.
doi:10.1107/S0907444910041004
PMCID: PMC3016015  PMID: 21206056
dihydrofolate reductase; inhibitors
4.  Multi-crystal anomalous diffraction for low-resolution macromolecular phasing 
Anomalous diffraction signals can be very weak and sensitive to radiation damage. Here, in application to a poorly diffracting (d min of 3.5 Å) and relatively large structure (1456 ordered residues), it is shown that data merged from multiple crystals can support SAD structure determination when no single data set is adequate.
Multiwavelength anomalous diffraction (MAD) and single-wavelength anomalous diffraction (SAD) are the two most commonly used methods for de novo determination of macromolecular structures. Both methods rely on the accurate extraction of anomalous signals; however, because of factors such as poor intrinsic order, radiation damage, inadequate anomalous scatterers, poor diffraction quality and other noise-causing factors, the anomalous signal from a single crystal is not always good enough for structure solution. In this study, procedures for extracting more accurate anomalous signals by merging data from multiple crystals are devised and tested. SAD phasing tests were made with a relatively large (1456 ordered residues) poorly diffracting (d min = 3.5 Å) selenomethionyl protein (20 Se). It is quantified that the anomalous signal, success in substructure determination and accuracy of phases and electron-density maps all improve with an increase in the number of crystals used in merging. Structure solutions are possible when no single crystal can support structural analysis. It is proposed that such multi-crystal strategies may be broadly useful when only weak anomalous signals are available.
doi:10.1107/S0907444910046573
PMCID: PMC3016016  PMID: 21206061
anomalous scattering; MAD; multiple crystals; phase determination; SAD
5.  Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding 
A new crystal form of the R163K variant of human thymidylate synthase (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181–197 in the active conformation, is reported.
Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181–197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed that hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.
doi:10.1107/S0907444910044732
PMCID: PMC3016017  PMID: 21206062
thymidylate synthase; asymmetry; glutathione; conformational switching; FdUMP; dUMP
6.  New clues in the allosteric activation of DNA cleavage by SgrAI: structures of SgrAI bound to cleaved primary-site DNA and uncleaved secondary-site DNA 
The structures of SgrAI bound to secondary-site DNA and Ca2+ or Mg2+, as well as bound to cleaved primary-site DNA and Mg2+, are presented and show similar overall conformations interpreted as the low-activity form of the enzyme. A third Mg2+ ion-binding site is found in the structure with cleaved primary-site DNA, as predicted by the two-metal-ion mechanism.
SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-activation with expansion of DNA-sequence specificity. The three-dimensional crystal structures of SgrAI bound to cleaved primary-site DNA and Mg2+ and bound to secondary-site DNA with either Mg2+ or Ca2+ are presented. All three structures show a conformation of enzyme and DNA similar to the previously determined dimeric structure of SgrAI bound to uncleaved primary-site DNA and Ca2+ [Dunten et al. (2008 ▶), Nucleic Acids Res. 36, 5405–5416], with the exception of the cleaved bond and a slight shifting of the DNA in the SgrAI/cleaved primary-site DNA/Mg2+ structure. In addition, a new metal ion binding site is located in one of the two active sites in this structure, which is consistent with proposals for the existence of a metal-ion site near the 3′-O leaving group.
doi:10.1107/S0907444910047785
PMCID: PMC3016018  PMID: 21206063
SgrAI; DNA cleavage; restriction endonucleases
7.  Preferential selection of isomer binding from chiral mixtures: alternate binding modes observed for the E and Z isomers of a series of 5-substituted 2,4-­diaminofuro[2,3-d]pyrimidines as ternary complexes with NADPH and human dihydrofolate reductase 
The structures of six chirally mixed E/Z-isomers of 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines reveals only one isomer is bound in the active site of human DHFR. The configuration of all but one C9-analogue is observed as the E-isomer.
The crystal structures of six human dihydrofolate reductase (hDHFR) ternary complexes with NADPH and a series of mixed E/Z isomers of 5-substituted 5-[2-(2-methoxyphenyl)-prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines substituted at the C9 position with propyl, isopropyl, cyclopropyl, butyl, isobutyl and sec-butyl (E2–E7, Z3) were determined and the results were compared with the resolved E and Z isomers of the C9-methyl parent compound. The configuration of all of the inhibitors, save one, was observed as the E isomer, in which the binding of the furopyrimidine ring is flipped such that the 4-­amino group binds in the 4-oxo site of folate. The Z3 isomer of the C9-isopropyl analog has the normal 2,4-diamino­pyrimidine ring binding geometry, with the furo oxygen near Glu30 and the 4-amino group interacting near the cofactor nicotinamide ring. Electron-density maps for these structures revealed the binding of only one isomer to hDHFR, despite the fact that chiral mixtures (E:Z ratios of 2:1, 3:1 and 3:2) of the inhibitors were incubated with hDHFR prior to crystallization. Superposition of the hDHFR com­plexes with E2 and Z3 shows that the 2′-methoxyphenyl ring of E2 is perpendicular to that of Z3. The most potent inhibitor in this series is the isopropyl analog Z3 and the least potent is the isobutyl analog E6, consistent with data that show that the Z isomer makes the most favorable interactions with the active-site residues. The isobutyl moiety of E6 is observed in two orientations and the resultant steric crowding of the E6 analog is consistent with its weaker activity. The alternative binding modes observed for the furopyrimidine ring in these E/Z isomers suggest that new templates can be designed to probe these binding regions of the DHFR active site.
doi:10.1107/S0907444910035808
PMCID: PMC2995722  PMID: 21123866
human dihydrofolate reductase; isomer binding
8.  The 2.7 Å resolution structure of the glycopeptide sulfotransferase Teg14 
The 2.7 Å resolution crystal structure of Teg14, a glycopeptide sulfotransferase cloned from an uncultured soil bacterium, is described. The relationship of Teg14 to other sulfotransferases is discussed.
The TEG gene cluster was recently isolated from an environmental DNA library and is predicted to encode the biosynthesis of a polysulfated glycopeptide congener. Three closely related sulfotransferases found in the TEG gene cluster (Teg12, Teg13 and Teg14) have been shown to sulfate the teicoplanin aglycone at three unique sites. Crystal structures of the first sulfotransferase from the TEG cluster, Teg12, in complex with the teicoplanin aglycone and its desulfated cosubstrate PAP have recently been reported [Bick et al. (2010 ▶), Biochemistry, 49, 4159–4168]. Here, the 2.7 Å resolution crystal structure of the apo form of Teg14 is reported. Teg14 sulfates the hydroxyphenylglycine at position 4 in the teicoplanin aglycone. The Teg14 structure is discussed and is compared with those of other bacterial 3′-phospho­adenosine 5′-­phosphosulfate-dependent sulfotransferases.
doi:10.1107/S0907444910036681
PMCID: PMC2995723  PMID: 21123867
glycopeptide sulfotransferases; Teg14
9.  Neutron structure and mechanistic studies of diisopropyl fluorophosphatase (DFPase) 
The structure and mechanism of diisopropyl fluorophosphatase (DFPase) have been studied using a variety of methods, including isotopic labelling, X-ray crystallography and neutron crystallography. The neutron structure of DFPase, mechanistic studies and subsequent rational design efforts are described.
Diisopropyl fluorophosphatase (DFPase) is a calcium-dependent phosphotriesterase that acts on a variety of highly toxic organophosphorus compounds that act as inhibitors of acetylcholinesterase. The mechanism of DFPase has been probed using a variety of methods, including isotopic labelling, which demonstrated the presence of a phosphoenzyme intermediate in the reaction mechanism. In order to further elucidate the mechanism of DFPase and to ascertain the protonation states of the residues and solvent molecules in the active site, the neutron structure of DFPase was solved at 2.2 Å resolution. The proposed nucleophile Asp229 is deprotonated, while the active-site solvent molecule W33 was identified as water and not hydroxide. These data support a mechanism involving direct nucleophilic attack by Asp229 on the substrate and rule out a mechanism involving metal-assisted water activation. These data also allowed for the re-engineering of DFPase through rational design to bind and productively orient the more toxic S P stereoisomers of the nerve agents sarin and cyclosarin, creating a modified enzyme with enhanced overall activity and significantly increased detoxification properties.
doi:10.1107/S0907444910034013
PMCID: PMC2967418  PMID: 21041927
neutron crystallography; DFPase; enzymes; rational design; phosphotriesterases; mechanism
10.  Protonation states of histidine and other key residues in deoxy normal human adult hemoglobin by neutron protein crystallography 
Using neutron diffraction analysis, the protonation states of 35 of 38 histidine residues were determined for the deoxy form of normal human adult hemoglobin. Distal and buried histidines may contribute to the increased affinity of the deoxy state for hydrogen ions and its decreased affinity for oxygen compared with the oxygenated form.
The protonation states of the histidine residues key to the function of deoxy (T-state) human hemoglobin have been investigated using neutron protein crystallography. These residues can reversibly bind protons, thereby regulating the oxygen affinity of hemoglobin. By examining the OMIT F o − F c and 2F o − F c neutron scattering maps, the protonation states of 35 of the 38 His residues were directly determined. The remaining three residues were found to be disordered. Surprisingly, seven pairs of His residues from equivalent α or β chains, αHis20, αHis50, αHis58, αHis89, βHis63, βHis143 and βHis146, have different protonation states. The proton­ation of distal His residues in the α1β1 heterodimer and the protonation of αHis103 in both subunits demonstrates that these residues may participate in buffering hydrogen ions and may influence the oxygen binding. The observed protonation states of His residues are compared with their ΔpK a between the deoxy and oxy states. Examination of inter-subunit interfaces provided evidence for interactions that are essential for the stability of the deoxy tertiary structure.
doi:10.1107/S0907444910025448
PMCID: PMC2967419  PMID: 21041929
human hemoglobin; deoxy form; protonation states; neutron protein crystallography; Bohr effect; histidine; deuterated water
11.  Joint X-ray and neutron refinement with phenix.refine  
The implementation of crystallographic structure-refinement procedures that include both X-ray and neutron data (separate or jointly) in the PHENIX system is described.
Approximately 85% of the structures deposited in the Protein Data Bank have been solved using X-ray crystallography, making it the leading method for three-dimensional structure determination of macromolecules. One of the limitations of the method is that the typical data quality (resolution) does not allow the direct determination of H-atom positions. Most hydrogen positions can be inferred from the positions of other atoms and therefore can be readily included into the structure model as a priori knowledge. However, this may not be the case in biologically active sites of macromolecules, where the presence and position of hydrogen is crucial to the enzymatic mechanism. This makes the application of neutron crystallo­graphy in biology particularly important, as H atoms can be clearly located in experimental neutron scattering density maps. Without exception, when a neutron structure is determined the corresponding X-ray structure is also known, making it possible to derive the complete structure using both data sets. Here, the implementation of crystallographic structure-refinement procedures that include both X-ray and neutron data (separate or jointly) in the PHENIX system is described.
doi:10.1107/S0907444910026582
PMCID: PMC2967420  PMID: 21041930
structure refinement; neutrons; joint X-ray and neutron refinement; PHENIX
12.  Enzymes for carbon sequestration: neutron crystallographic studies of carbonic anhydrase 
The first neutron crystal structure of carbonic anhydrase is presented. The structure reveals interesting and unexpected features of the active site that affect catalysis.
Carbonic anhydrase (CA) is a ubiquitous metalloenzyme that catalyzes the reversible hydration of CO2 to form HCO3 − and H+ using a Zn–hydroxide mechanism. The first part of catalysis involves CO2 hydration, while the second part deals with removing the excess proton that is formed during the first step. Proton transfer (PT) is thought to occur through a well ordered hydrogen-bonded network of waters that stretches from the metal center of CA to an internal proton shuttle, His64. These waters are oriented and ordered through a series of hydrogen-bonding interactions to hydrophilic residues that line the active site of CA. Neutron studies were conducted on wild-type human CA isoform II (HCA II) in order to better understand the nature and the orientation of the Zn-bound solvent (ZS), the charged state and conformation of His64, the hydrogen-bonding patterns and orientations of the water molecules that mediate PT and the ionization of hydrophilic residues in the active site that interact with the water network. Several interesting and unexpected features in the active site were observed which have implications for how PT proceeds in CA.
doi:10.1107/S0907444910019700
PMCID: PMC2967421  PMID: 21041933
proton transfer; deprotonated tyrosine; carbonic anhydrase; neutron crystallography
13.  Macromolecular neutron crystallography at the Protein Crystallography Station (PCS) 
The Protein Crystallography Station user facility at Los Alamos National Laboratory not only offers open access to a high-performance neutron beamline, but also actively supports and develops new methods in protein expression, deuteration, purification, robotic crystallization and the synthesis of substrates with stable isotopes and provides assistance with data-reduction and structure-refinement software and comprehensive neutron structure analysis.
The Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. Neutron diffraction is a powerful technique for locating H atoms and can therefore provide unique information about how biological macro­molecules function and interact with each other and smaller molecules. Users of the PCS have access to neutron beam time, deuteration facilities, the expression of proteins and the synthesis of substrates with stable isotopes and also support for data reduction and structure analysis. The beamline exploits the pulsed nature of spallation neutrons and a large electronic detector in order to collect wavelength-resolved Laue patterns using all available neutrons in the white beam. The PCS user facility is described and highlights from the user program are presented.
doi:10.1107/S0907444910027198
PMCID: PMC2967422  PMID: 21041938
Protein Crystallography Station; neutron macromolecular crystallography; spallation neutron sources; deuteration; user support
14.  Hemoglobin redux: combining neutron and X-ray diffraction with mass spectrometry to analyse the quaternary state of oxidized hemoglobins 
X-ray and neutron diffraction studies of cyanomethemoglobin are being used to evaluate the structural waters within the dimer–dimer interface involved in quaternary-state transitions.
Improvements in neutron diffraction instrumentation are affording the opportunity to re-examine the structures of vertebrate hemoglobins and to interrogate proton and solvent position changes between the different quaternary states of the protein. For hemoglobins of unknown primary sequence, structural studies of cyanomethemoglobin (CNmetHb) are being used to help to resolve sequence ambiguity in the mass spectra. These studies have also provided additional structural evidence for the involvement of oxidized hemoglobin in the process of erythrocyte senescence. X-ray crystal studies of Tibetan snow leopard CNmetHb have shown that this protein crystallizes in the B state, a structure with a more open dyad, which possibly has relevance to RBC band 3 protein binding and erythrocyte senescence. R-state equine CNmetHb crystal studies elaborate the solvent differences in the switch and hinge region compared with a human deoxyhemoglobin T-­state neutron structure. Lastly, comparison of histidine protonation between the T and R state should enumerate the Bohr-effect protons.
doi:10.1107/S090744491002545X
PMCID: PMC2967423  PMID: 21041946
equine hemoglobin; snow leopard hemoglobin; mass spectrometry; protein sequencing; time-of-flight neutron diffraction; R state; T state; B state; joint XN refinement; protonation state
15.  Using neutron protein crystallography to understand enzyme mechanisms 
A description is given of the results of neutron diffraction studies of the structures of four different metal-ion complexes of deuterated d-xylose isomerase.
A description is given of the results of neutron diffraction studies of the structures of four different metal-ion complexes of deuterated d-xylose isomerase. These represent four stages in the progression of the biochemical catalytic action of this enzyme. Analyses of the structural changes observed between the various three-dimensional structures lead to some insight into the mechanism of action of this enzyme.
doi:10.1107/S0907444910027915
PMCID: PMC2967424  PMID: 21041947
neutron diffraction; enzyme mechanisms; d-xylose isomerase
16.  Glass transition in thaumatin crystals revealed through temperature-dependent radiation-sensitivity measurements 
Radiation damage to protein crystals exhibits two regimes of temperature-activated behavior between T = 300 and 100 K, with a crossover at the protein glass transition near 200 K. These results have implications for mechanistic studies of proteins and for structure determination when cooling to T = 100 K creates excessive disorder.
The temperature-dependence of radiation damage to thaumatin crystals between T = 300 and 100 K is reported. The amount of damage for a given dose decreases sharply as the temperature decreases from 300 to 220 K and then decreases more gradually on further cooling below the protein-solvent glass transition. Two regimes of temperature-activated behavior were observed. At temperatures above ∼200 K the activation energy of 18.0 kJ mol−1 indicates that radiation damage is dominated by diffusive motions in the protein and solvent. At temperatures below ∼200 K the activation energy is only 1.00 kJ mol−1, which is of the order of the thermal energy. Similar activation energies describe the temperature-dependence of radiation damage to a variety of solvent-free small-molecule organic crystals over the temperature range T = 300–80 K. It is suggested that radiation damage in this regime is vibrationally assisted and that the freezing-out of amino-acid scale vibrations contributes to the very weak temperature-dependence of radiation damage below ∼80 K. Analysis using the radiation-damage model of Blake and Phillips [Blake & Phillips (1962 ▶), Biological Effects of Ionizing Radiation at the Molecular Level, pp. 183–191] indicates that large-scale conformational and molecular motions are frozen out below T = 200 K but become increasingly prevalent and make an increasing contribution to damage at higher temperatures. Possible alternative mechanisms for radiation damage involving the formation of hydrogen-gas bubbles are discussed and discounted. These results have implications for mechanistic studies of proteins and for studies of the protein glass transition. They also suggest that data collection at T ≃ 220 K may provide a viable alternative for structure determination when cooling-induced disorder at T = 100 is excessive.
doi:10.1107/S0907444910035523
PMCID: PMC2954455  PMID: 20944242
protein crystallography; radiation damage; temperature dependence; glass transition
17.  Binding of flexible and constrained ligands to the Grb2 SH2 domain: structural effects of ligand preorganization 
Structures of the Grb2 SH2 domain complexed with a series of flexible and constrained replacements of the phosphotyrosine residue in tripeptides derived from Ac-pYXN (where X = V, I, E and Q) were compared to determine what, if any, structural differences arise as a result of ligand preorganization.
Structures of the Grb2 SH2 domain complexed with a series of pseudopeptides containing flexible (benzyl succinate) and constrained (aryl cyclopropanedicarboxylate) replacements of the phosphotyrosine (pY) residue in tripeptides derived from Ac-pYXN-NH2 (where X = V, I, E and Q) were elucidated by X-ray crystallography. Complexes of flexible/constrained pairs having the same pY + 1 amino acid were analyzed in order to ascertain what structural differences might be attributed to constraining the phosphotyrosine replacement. In this context, a given structural dissimilarity between complexes was considered to be significant if it was greater than the corresponding difference in complexes coexisting within the same asymmetric unit. The backbone atoms of the domain generally adopt a similar conformation and orientation relative to the ligands in the complexes of each flexible/constrained pair, although there are some significant differences in the relative orientations of several loop regions, most notably in the BC loop that forms part of the binding pocket for the phosphate group in the tyrosine replacements. These variations are greater in the set of complexes of constrained ligands than in the set of complexes of flexible ligands. The constrained ligands make more direct polar contacts to the domain than their flexible counterparts, whereas the more flexible ligand of each pair makes more single-water-mediated contacts to the domain; there was no correlation between the total number of protein–ligand contacts and whether the phosphotyrosine replacement of the ligand was preorganized. The observed differences in hydrophobic interactions between the complexes of each flexible/constrained ligand pair were generally similar to those observed upon comparing such contacts in coexisting complexes. The average adjusted B factors of the backbone atoms of the domain and loop regions are significantly greater in the complexes of constrained ligands than in the complexes of the corresponding flexible ligands, suggesting greater thermal motion in the crystalline state in the former complexes. There was no apparent correlation between variations in crystal packing and observed structural differences or similarities in the complexes of flexible and constrained ligands, but the possibility that crystal packing might result in structural variations cannot be rigorously excluded. Overall, it appears that there are more variations in the three-dimensional structure of the protein and the ligand in complexes of the constrained ligands than in those of their more flexible counterparts.
doi:10.1107/S0907444910035584
PMCID: PMC2954456  PMID: 20944243
Grb2 SH2; protein–ligand interactions; constraints; preorganization
18.  Structure of the N-terminal fragment of Escherichia coli Lon protease 
The medium-resolution structure of the N-terminal fragment of E. coli Lon protease shows that this part of the enzyme consists of two compact domains and a very long α-helix.
The structure of a recombinant construct consisting of residues 1–245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.
doi:10.1107/S0907444910019554
PMCID: PMC2917273  PMID: 20693685
anomalous diffraction; ATP-dependent proteases; protein domains; structure quality; Lon protease
19.  Validation of crystallographic models containing TLS or other descriptions of anisotropy 
Guidelines and specific tests for validating macromolecular crystal structures that include TLS models are introduced. Validation may used to troubleshoot problems during refinement, to confirm the internal consistency of the model as part of deposition into the Protein Data Bank or to assess the plausibility of interpretating the boundary between two TLS groups as indicating a hinge point between structural domains.
The use of TLS (translation/libration/screw) models to describe anisotropic displacement of atoms within a protein crystal structure has become increasingly common. These models may be used purely as an improved methodology for crystallographic refinement or as the basis for analyzing inter-domain and other large-scale motions implied by the crystal structure. In either case it is desirable to validate that the crystallographic model, including the TLS description of anisotropy, conforms to our best understanding of protein structures and their modes of flexibility. A set of validation tests has been implemented that can be integrated into ongoing crystallographic refinement or run afterwards to evaluate a previously refined structure. In either case validation can serve to increase confidence that the model is correct, to highlight aspects of the model that may be improved or to strengthen the evidence supporting specific modes of flexibility inferred from the refined TLS model. Automated validation checks have been added to the PARVATI and TLSMD web servers and incorporated into the CCP4i user interface.
doi:10.1107/S0907444910020421
PMCID: PMC2917275  PMID: 20693688
validation; TLS models; anisotropy
20.  Enhancing MAD F A data for substructure determination 
A new statistical and computational procedure, which merges multiple F A estimates into an averaged data set, is used to further improve the quality of the estimated anomalous amplitudes for substructure determination.
Heavy-atom substructure determination is a critical step in phasing an unknown macromolecular structure. Dual-space (Shake-and-Bake) recycling is a very effective procedure for locating the substructure (heavy) atoms using F A data estimated from multiple-wavelength anomalous diffraction. However, the estimated F A are susceptible to the accumulation of errors in the individual intensity measurements at several wavelengths and from inaccurate estimation of the anomalous atomic scattering corrections f′ and f′′. In this paper, a new statistical and computational procedure which merges multiple F A estimates into an averaged data set is used to further improve the quality of the estimated anomalous amplitudes. The results of 18 Se-atom substructure determinations provide convincing evidence in favor of using such a procedure to locate anomalous scatterers.
doi:10.1107/S0907444910025783
PMCID: PMC2917277  PMID: 20693694
substructure determination; MAD data
21.  Emerging from pseudo-symmetry: the redetermination of human carbonic anhydrase II in monoclinic P21 with a doubled a axis 
The structure of human carbonic anhydrase II in the monoclinic P21 space group with a doubled a axis from that of the usually observed unit cell has been re-determined and shown that the choice for how the four molecules in the unit cell are grouped (based on their coordinates) into pairs that represent a single asymmetric unit determines whether or not rotational disorder is observed/created during refinement.
The crystal structure of human carbonic anhydrase II in the monoclinic P21 space group with a doubled a axis from that of the usually observed unit cell has recently been reported, with one of the two molecules in the asymmetric unit demonstrating rotational disorder [Robbins et al. (2010 ▶), Acta Cryst. D66, 628–634]. The structure has been redetermined, with the coordinates of both pseudo-symmetrically related molecules in the crystallographic asymmetric unit translated by x′ = x ± 1/4, and no rotational disorder is observed. This corresponds to a different choice of how the four molecules in the unit cell should be grouped into pairs that represent a single asymmetric unit.
doi:10.1107/S0907444910023723
PMCID: PMC2917278  PMID: 20693695
doubled axis; systematically weak data; pseudo-translational symmetry; redetermination
22.  Structure of the Escherichia coli RNA polymerase α subunit C-terminal domain 
The crystal structure of the dimethyllysine derivative of the E. coli RNA polymerase α subunit C-terminal domain is reported at 2.0 Å resolution.
The α subunit C-terminal domain (αCTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli αCTD (α subunit residues 245–329) determined to 2.0 Å resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P21 and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, R free = 0.236) has improved geometry compared with prior lower resolution determinations of the αCTD structure [Jeon et al. (1995 ▶), Science, 270, 1495–1497; Benoff et al. (2002 ▶), Science, 297, 1562–1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of αCTD-containing multi-component complexes visualized at lower resolution using X-ray crystallo­graphy and electron-microscopy reconstruction.
doi:10.1107/S0907444910018470
PMCID: PMC2897699  PMID: 20606261
RNA polymerase; Escherichia coli; α subunit; C-terminal domain
23.  Using a conformation-dependent stereochemical library improves crystallographic refinement of proteins 
A stereochemical library which defines the target values for main-chain bond lengths and angles as a function of the residue’s ϕ/ψ angles was tested in refinement. Use of this library allows the construction of models that conform to ideal geometry much better than previous libraries without degrading their fit to the diffraction data.
The major macromolecular crystallographic refinement packages restrain models to ideal geometry targets defined as single values that are independent of molecular conformation. However, ultrahigh-resolution X-ray models of proteins are not consistent with this concept of ideality and have been used to develop a library of ideal main-chain bond lengths and angles that are parameterized by the ϕ/ψ angle of the residue [Berkholz et al. (2009 ▶), Structure, 17, 1316–1325]. Here, it is first shown that the new conformation-dependent library does not suffer from poor agreement with ultrahigh-resolution structures, whereas current libraries have this problem. Using the TNT refinement package, it is then shown that protein structure refinement using this conformation-dependent library results in models that have much better agreement with library values of bond angles with little change in the R values. These tests support the value of revising refinement software to account for this new paradigm.
doi:10.1107/S0907444910019207
PMCID: PMC2897700  PMID: 20606264
conformation-dependent stereochemical library; refinement; ideal geometry; restraints
24.  Structure of the d-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes 
The structure of EhpF from P. agglomerans has been solved alone and in complex with phenazine-1,6-dicarboxylate. Apo EhpF was solved and refined in two different space groups at 1.95 and 2.3 Å resolution and the EhpF–phenazine-1,6-dicarboxylate complex structure was determined at 2.8 Å resolution.
The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound d-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-­carboxylate via an adenylyl intermediate.
doi:10.1107/S0907444910008425
PMCID: PMC2879354  PMID: 20516619
chorismate; d-alanylgriseoluteic acid; phenazine-1,6-dicarboxylic acid
25.  Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions 
The first structure of tartrate dehydrogenase, solved as an intermediate analog complex, allows identification of the substrate-binding and catalytic functional groups. Roles are confirmed for the required monovalent and divalent cations, and intersubunit communication channels are proposed in support of an alternating-site reaction mechanism.
The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 Å resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg2+ and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. How­ever, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate inter­mediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.
doi:10.1107/S0907444910008851
PMCID: PMC2879355  PMID: 20516620
tartrate dehydrogenase; Pseudomonas putida; hydroxy-acid dehydrogenases

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