The structure of the rat vitamin B12 transporter TC and its complex with glutathionylcobalamin.

Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B12. Currently, there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12 transporter and GSCbl, which compared to aquo/hydroxocobalamin, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, haptocorrin and intrinsic factor are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl.

Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases.

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.

Short-lived intermediate in N2O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography.

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N2O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate ( I ), a key state to promote N-N bond formation and N-O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe-NO coordination in I , with an elongated Fe-NO bond length (Fe-NO = 1.91 Å, Fe-N-O = 138°) in the absence of NAD+ TR-infrared (IR) spectroscopy detects the formation of I with an N-O stretching frequency of 1,290 cm-1 upon hydride transfer from NADH to the Fe3+-NO enzyme via the dissociation of NAD+ from a transient state, with an N-O stretching of 1,330 cm-1 and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe3+-NHO•- radical. The current findings provide conclusive evidence for the N2O generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule.

Crystal structure of a nucleotide-binding domain of fatty acid kinase FakA from Thermus thermophilus HB8.

Fatty acid kinase is necessary for the incorporation of exogenous fatty acids into membrane phospholipids. Fatty acid kinase consists of two components: a kinase component, FakA, that phosphorylates a fatty acid bound to a fatty acid-binding component, FakB. However, the molecular details underlying the phosphotransfer reaction remain to be resolved. We determined the crystal structure of the N-terminal domain of FakA bound to ADP from Thermus thermophilus HB8. The overall structure of this domain showed that the helical barrel fold is similar to the nucleotide-binding component of dihydroxyacetone kinase. The structure of the nucleotide-binding site revealed the roles of the conserved residues in recognition of ADP and Mg2+, but the N-terminal domain of FakA lacked the ADP-capping loop found in the dihydroxyacetone kinase component. Based on the structural similarity to the two subunits of dihydroxyacetone kinase complex, we constructed a model of the complex of T. thermophilus FakB and the N-terminal domain of FakA. In this model, the invariant Arg residue of FakB occupied a position that was spatially similar to that of the catalytically important Arg residue of dihydroxyacetone kinase, which predicted a composite active site in the Fatty acid kinase complex.

Guidelines for de novo phasing using multiple small-wedge data collection. Corrigendum.

A figure in the article by Baba et al. [(2021), J. Synchrotron Rad. 28, 1284-1295] is corrected.

Self-assembly of tetravalent Goldberg polyhedra from 144 small components.

Rational control of the self-assembly of large structures is one of the key challenges in chemistry, and is believed to become increasingly difficult and ultimately impossible as the number of components involved increases. So far, it has not been possible to design a self-assembled discrete molecule made up of more than 100 components. Such molecules-for example, spherical virus capsids-are prevalent in nature, which suggests that the difficulty in designing these very large self-assembled molecules is due to a lack of understanding of the underlying design principles. For example, the targeted assembly of a series of large spherical structures containing up to 30 palladium ions coordinated by up to 60 bent organic ligands was achieved by considering their topologies. Here we report the self-assembly of a spherical structure that also contains 30 palladium ions and 60 bent ligands, but belongs to a shape family that has not previously been observed experimentally. The new structure consists of a combination of 8 triangles and 24 squares, and has the symmetry of a tetravalent Goldberg polyhedron. Platonic and Archimedean solids have previously been prepared through self-assembly, as have trivalent Goldberg polyhedra, which occur naturally in the form of virus capsids and fullerenes. But tetravalent Goldberg polyhedra have not previously been reported at the molecular level, although their topologies have been predicted using graph theory. We use graph theory to predict the self-assembly of even larger tetravalent Goldberg polyhedra, which should be more stable, enabling another member of this polyhedron family to be assembled from 144 components: 48 palladium ions and 96 bent ligands.

In crystallo thermodynamic analysis of conformational change of the topaquinone cofactor in bacterial copper amine oxidase.

In the catalytic reaction of copper amine oxidase, the protein-derived redox cofactor topaquinone (TPQ) is reduced by an amine substrate to an aminoresorcinol form (TPQamr), which is in equilibrium with a semiquinone radical (TPQsq). The transition from TPQamr to TPQsq is an endothermic process, accompanied by a significant conformational change of the cofactor. We employed the humid air and glue-coating (HAG) method to capture the equilibrium mixture of TPQamr and TPQsq in noncryocooled crystals of the enzyme from Arthrobacter globiformis and found that the equilibrium shifts more toward TPQsq in crystals than in solution. Thermodynamic analyses of the temperature-dependent equilibrium also revealed that the transition to TPQsq is entropy-driven both in crystals and in solution, giving the thermodynamic parameters that led to experimental determination of the crystal packing effect. Furthermore, we demonstrate that the binding of product aldehyde to the hydrophobic pocket in the active site produces various equilibrium states among two forms of the product Schiff-base, TPQamr, and TPQsq, in a pH-dependent manner. The temperature-controlled HAG method provides a technique for thermodynamic analysis of conformational changes occurring in protein crystals that are hardly scrutinized by conventional cryogenic X-ray crystallography.

A Lynch syndrome-associated mutation at a Bergerat ATP-binding fold destabilizes the structure of the DNA mismatch repair endonuclease MutL.

In humans, mutations in genes encoding homologs of the DNA mismatch repair endonuclease MutL cause a hereditary cancer that is known as Lynch syndrome. Here, we determined the crystal structures of the N-terminal domain (NTD) of MutL from the thermophilic eubacterium Aquifex aeolicus (aqMutL) complexed with ATP analogs at 1.69-1.73 Å. The structures revealed significant structural similarities to those of a human MutL homolog, postmeiotic segregation increased 2 (PMS2). We introduced five Lynch syndrome-associated mutations clinically found in human PMS2 into the aqMutL NTD and investigated the protein stability, ATPase activity, and DNA-binding ability of these protein variants. Among the mutations studied, the most unexpected results were obtained for the residue Ser34. Ser34 (Ser46 in PMS2) is located at a previously identified Bergerat ATP-binding fold. We found that the S34I aqMutL NTD retains ATPase and DNA-binding activities. Interestingly, CD spectrometry and trypsin-limited proteolysis indicated the disruption of a secondary structure element of the S34I NTD, destabilizing the overall structure of the aqMutL NTD. In agreement with this, the recombinant human PMS2 S46I NTD was easily digested in the host Escherichia coli cells. Moreover, other mutations resulted in reduced DNA-binding or ATPase activity. In summary, using the thermostable aqMutL protein as a model molecule, we have experimentally determined the effects of the mutations on MutL endonuclease; we discuss the pathological effects of the corresponding mutations in human PMS2.

Oncogenic mutations Q61L and Q61H confer active form-like structural features to the inactive state (state 1) conformation of H-Ras protein.

GTP-bound forms of Ras proteins (Ras•GTP) assume two interconverting conformations, "inactive" state 1 and "active" state 2. Our previous study on the crystal structure of the state 1 conformation of H-Ras in complex with guanosine 5'-(ß, γ-imido)triphosphate (GppNHp) indicated that state 1 is stabilized by intramolecular hydrogen-bonding interactions formed by Gln61. Since Ras are constitutively activated by substitution mutations of Gln61, here we determine crystal structures of the state 1 conformation of H-Ras•GppNHp carrying representative mutations Q61L and Q61H to observe the effect of the mutations. The results show that these mutations alter the mode of hydrogen-bonding interactions of the residue 61 with Switch II residues and induce conformational destabilization of the neighboring regions. In particular, Q61L mutation results in acquirement of state 2-like structural features. Moreover, the mutations are likely to impair an intramolecular structural communication between Switch I and Switch II. Molecular dynamics simulations starting from these structures support the above observations. These findings may give a new insight into the molecular mechanism underlying the aberrant activation of the Gln61 mutants.

Guidelines for de novo phasing using multiple small-wedge data collection.

Intense micro-focus X-ray beamlines available at synchrotron facilities have achieved high-quality data collection even from the microcrystals of membrane proteins. The automatic data collection system developed at SPring-8, named ZOO, has contributed to many structure determinations of membrane proteins using small-wedge synchrotron crystallography (SWSX) datasets. The `small-wedge' (5-20°) datasets are collected from multiple crystals and then merged to obtain the final structure factors. To our knowledge, no systematic investigation on the dose dependence of data accuracy has so far been reported for SWSX, which is between `serial crystallography' and `rotation crystallography'. Thus, herein, we investigated the optimal dose conditions for experimental phasing with SWSX. Phase determination using anomalous scattering signals was found to be more difficult at higher doses. Furthermore, merging more homogeneous datasets grouped by hierarchical clustering with controlled doses mildly reduced the negative factors in data collection, such as `lack of signal' and `radiation damage'. In turn, as more datasets were merged, more probable phases could be obtained across a wider range of doses. Therefore, our findings show that it is essential to choose a lower dose than 10 MGy for de novo structure determination by SWSX. In particular, data collection using a dose of 5 MGy proved to be optimal in balancing the amount of signal available while reducing the amount of damage as much as possible.

Analysis of oscillatory rocking curve by dynamical diffraction in protein crystals.

High-quality protein crystals meant for structural analysis by X-ray diffraction have been grown by various methods. The observation of dynamical diffraction in protein crystals is an interesting topic because dynamical diffraction generally occurs in perfect crystals such as Si crystals. However, to our knowledge, there is no report yet on protein crystals showing clear dynamical diffraction. We wonder whether the perfection of protein crystals might still be low compared with that of high-quality Si crystals. Here, we present observations of the oscillatory profile of rocking curves for protein crystals such as glucose isomerase crystals. The oscillatory profiles are in good agreement with those predicted by the dynamical theory of diffraction. We demonstrate that dynamical diffraction occurs even in protein crystals. This suggests the possibility of the use of dynamical diffraction for the determination of the structure and charge density of proteins.

Low-dose X-ray structure analysis of cytochrome c oxidase utilizing high-energy X-rays.

To investigate the effect of high-energy X-rays on site-specific radiation-damage, low-dose diffraction data were collected from radiation-sensitive crystals of the metal enzyme cytochrome c oxidase. Data were collected at the Structural Biology I beamline (BL41XU) at SPring-8, using 30 keV X-rays and a highly sensitive pixel array detector equipped with a cadmium telluride sensor. The experimental setup of continuous sample translation using multiple crystals allowed the average diffraction weighted dose per data set to be reduced to 58 kGy, and the resulting data revealed a ligand structure featuring an identical bond length to that in the damage-free structure determined using an X-ray free-electron laser. However, precise analysis of the residual density around the ligand structure refined with the synchrotron data showed the possibility of a small level of specific damage, which might have resulted from the accumulated dose of 58 kGy per data set. Further investigation of the photon-energy dependence of specific damage, as assessed by variations in UV-vis absorption spectra, was conducted using an on-line spectrometer at various energies ranging from 10 to 30 keV. No evidence was found for specific radiation damage being energy dependent.

A solute-binding protein in the closed conformation induces ATP hydrolysis in a bacterial ATP-binding cassette transporter involved in the import of alginate.

The Gram-negative bacterium Sphingomonas sp. A1 incorporates alginate into cells via the cell-surface pit without prior depolymerization by extracellular enzymes. Alginate import across cytoplasmic membranes thereby depends on the ATP-binding cassette transporter AlgM1M2SS (a heterotetramer of AlgM1, AlgM2, and AlgS), which cooperates with the periplasmic solute-binding protein AlgQ1 or AlgQ2; however, several details of AlgM1M2SS-mediated alginate import are not well-understood. Herein, we analyzed ATPase and transport activities of AlgM1M2SS after reconstitution into liposomes with AlgQ2 and alginate oligosaccharide substrates having different polymerization degrees (PDs). Longer alginate oligosaccharides (PD ≥ 5) stimulated the ATPase activity of AlgM1M2SS but were inert as substrates of AlgM1M2SS-mediated transport, indicating that AlgM1M2SS-mediated ATP hydrolysis can be stimulated independently of substrate transport. Using X-ray crystallography in the presence of AlgQ2 and long alginate oligosaccharides (PD 6-8) and with the humid air and glue-coating method, we determined the crystal structure of AlgM1M2SS in complex with oligosaccharide-bound AlgQ2 at 3.6 Å resolution. The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solute-binding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. These in vitro assays and structural analyses indicated that interactions between AlgM1M2SS in the inward-facing conformation and periplasmic ligand-bound AlgQ2 in the closed conformation induce ATP hydrolysis by the ATP-binding protein AlgS. We conclude that substrate-bound AlgQ2 in the closed conformation initially interacts with AlgM1M2SS, the AlgM1M2SS-AlgQ2 complex then forms, and this formation is followed by ATP hydrolysis.

Unpaired Electron Spin Density Distribution across Reduced [2Fe-2S] Cluster Ligands by 13Cß-Cysteine Labeling.

Iron-sulfur clusters are one of the most versatile and ancient classes of redox mediators in biology. The roles that these metal centers take on are predominantly determined by the number and types of coordinating ligands (typically cysteine and histidine) that modify the electronic structure of the cluster. Here we map the spin density distribution onto the cysteine ligands for the three major classes of the protein-bound, reduced [2Fe-2S](His)n(Cys)4-n (n = 0, 1, 2) cluster by selective cysteine-13Cß isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe2+ ligands than the nonreducible Fe3+ ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe2+ ligands is consistent with the structural concept that the orientation of the cluster in proteins is not arbitrarily decided, but rather is optimized such that it is likely to facilitate better electronic coupling with redox partners. The resolution of all cysteine-13Cß hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal-thiolate bonds not readily available to other techniques.

Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel.

Voltage-gated sodium (Na(v)) channels are essential for the rapid depolarization of nerve and muscle, and are important drug targets. Determination of the structures of Na(v) channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Na(v) channels, exemplified by the Na(+)-selective channel of bacteria (NaChBac), provides a useful model system for structure-function analysis. Here we report the crystal structure of Na(v)Rh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 (Rickettsiales sp. HIMB114; denoted Rh), at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr 178 and Leu 179 constitute an inner site within the selectivity filter where a hydrated Ca(2+) resides in the crystal structure. The outer mouth of the Na(+) selectivity filter, defined by Ser 181 and Glu 183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that Na(v)Rh is in an 'inactivated' conformation. Comparison of Na(v)Rh with Na(v)Ab reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

Structural Features and Functional Dependency on ß-Clamp Define Distinct Subfamilies of Bacterial Mismatch Repair Endonuclease MutL.

In early reactions of DNA mismatch repair, MutS recognizes mismatched bases and activates MutL endonuclease to incise the error-containing strand of the duplex. DNA sliding clamp is responsible for directing the MutL-dependent nicking to the newly synthesized/error-containing strand. In Bacillus subtilis MutL, the ß-clamp-interacting motif (ß motif) of the C-terminal domain (CTD) is essential for both in vitro direct interaction with ß-clamp and in vivo repair activity. A large cluster of negatively charged residues on the B. subtilis MutL CTD prevents nonspecific DNA binding until ß clamp interaction neutralizes the negative charge. We found that there are some bacterial phyla whose MutL endonucleases lack the ß motif. For example, the region corresponding to the ß motif is completely missing in Aquifex aeolicus MutL, and critical amino acid residues in the ß motif are not conserved in Thermus thermophilus MutL. We then revealed the 1.35 Å-resolution crystal structure of A. aeolicus MutL CTD, which lacks the ß motif but retains the metal-binding site for the endonuclease activity. Importantly, there was no negatively charged cluster on its surface. It was confirmed that CTDs of ß motif-lacking MutLs, A. aeolicus MutL and T. thermophilus MutL, efficiently incise DNA even in the absence of ß-clamp and that ß-clamp shows no detectable enhancing effect on their activity. In contrast, CTD of Streptococcus mutans, a ß motif-containing MutL, required ß-clamp for the digestion of DNA. We propose that MutL endonucleases are divided into three subfamilies on the basis of their structural features and dependence on ß-clamp.

Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography.

Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines. Unlike SFX, the longer exposure time of milliseconds to seconds commonly used in SSX causes radiation damage. However, in SSX, crystals can be rotated during the exposure, which can achieve efficient coverage of the reciprocal space. In this study, mercury single-wavelength anomalous diffraction (Hg-SAD) phasing of the luciferin regenerating enzyme (LRE) was performed using serial synchrotron rotation crystallography. The advantages of rotation and influence of dose on the data collected were evaluated. The results showed that sample rotation was effective for accurate data collection, and the optimum helical rotation step depended on multiple factors such as multiplicity and partiality of reflections, exposure time per rotation angle and the contribution from background scattering. For the LRE microcrystals, 0.25° was the best rotation step for the achievable resolution limit, whereas a rotation step larger than or equal to 1° was favorable for Hg-SAD phasing. Although an accumulated dose beyond 1.1 MGy caused specific damage at the Hg site, increases in resolution and anomalous signal were observed up to 3.4 MGy because of a higher signal-to-noise ratio.

Crystal structure and DNA-binding property of the ATPase domain of bacterial mismatch repair endonuclease MutL from Aquifex aeolicus.

DNA mismatch repair (MMR) system corrects mismatched bases that are generated mainly by DNA replication errors. The repair system excises the error-containing single-stranded region and enables the re-synthesis of the strand. In the early reactions of MMR, MutL endonuclease incises the newly-synthesized/error-containing strand of the duplex to initiate the downstream excision reaction. MutL endonuclease consists of the N-terminal ATPase and C-terminal endonuclease domains. In this study, we report the crystal structure of the ATPase domain of MutL endonuclease from Aquifex aeolicus. The overall structure of the domain was similar to those of human MutL homologs and Escherichia coli MutL, although E. coli MutL has no endonuclease activity. The ATPase domain was comprised of two subdomains: the N-terminal ATP-binding subdomain and the C-terminal α-ß sandwich subdomain. Site-directed mutagenesis experiment identified DNA-interacting eight basic amino acid residues, which were distributed across both the two subdomains and formed a DNA-binding cleft. Docking simulation between the structures of the ATPase and endonuclease domains generated a reliable model structure for the full-length A. aeolicus MutL, which satisfies our previous result of small-angle X-ray scattering analysis. On the basis of the model structure and further experimental results, we concluded that the two separate DNA-binding sites in the full-length A. aeolicus MutL simultaneously bind a dsDNA molecule.

In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction.

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.

Structure of the RsbX phosphatase involved in the general stress response of Bacillus subtilis.

In the general stress response of Bacillus subtilis, which is governed by the sigma factor σ(B), stress signalling is relayed by a cascade of Rsb proteins that regulate σ(B) activity. RsbX, a PPM II phosphatase, halts the response by dephosphorylating the stressosome composed of RsbR and RsbS. The crystal structure of RsbX reveals a reorganization of the catalytic centre, with the second Mn(2+) ion uniquely coordinated by Gly47 O from the ß4-α1 loop instead of a water molecule as in PPM I phosphatases. An extra helical turn of α1 tilts the loop towards the metal-binding site, and the ß2-ß3 loop swings outwards to accommodate this tilting. The residues critical for this defining feature of the PPM II phosphatases are highly conserved. Formation of the catalytic centre is metal-specific, as crystallization with Mg(2+) ions resulted in a shift of the ß4-α1 loop that led to loss of the second ion. RsbX also lacks the flap subdomain characteristic of PPM I phosphatases. On the basis of a stressosome model, the activity of RsbX towards RsbR-P and RsbS-P may be influenced by the different accessibilities of their phosphorylation sites.