Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating

Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca²⁺ can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the C^α atom of Glu31 10 Å away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating.     

Crystal Structure of a Yeast Aquaporin at 1.15 Å Reveals a Novel Gating Mechanism

Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.     

Crystal Structure of a Yeast Aquaporin at 1.15 ? Reveals a Novel Gating Mechanism

Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.     

X-ray crystal structure of the serine proteinase inhibitor eglin c at 1.95 A resolution.

The crystal structure of eglin c, naturally occurring in the leech Hirudo medicinalis, is known from its complexes with various serine proteinases, but the crystallization of free eglin c has not yet been reported. A method is described for growing well-diffracting crystals of free eglin c from highly concentrated protein solutions (approximately 200 mg/ml). The space group of the orthorhombic crystals was determined to be P2(1)2(1)2(1) with unit cell parameters a = 32.6, b = 42.0, c = 44.1 A. The structure of free eglin c was resolved at 1.95 A resolution by Patterson search methods. The final model contains all 70 amino acids of eglin c and 125 water molecules. In comparison to the eglin structure known from its complexes with proteinases, only small differences have been observed in free eglin c. However, the reactive site-binding loop and a few residues on the surface of eglin have been found in different conformations due to crystal contacts. In contrast to the complex structures, the first seven amino acids of the highly flexible amino terminus can be located. Crystallographic refinement comprised molecular dynamics refinement, classical restrained least-squares refinement and individual isotropic atomic temperature refinement. The final R-factor is 15.8%.     

Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

Aquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca²⁺-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg²⁺-dependent, Ca²⁺-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1.     

Identification and structure of a putative Ca2+-binding domain at the C terminus of AQP1

Aquaporin-1 (AQP1) is the first functionally identified aquaporin of a growing family of membrane water channels found in all forms of life. Recently, a possible secondary function as a cyclic guanosine monophosphate (cGMP) gated ion channel was attributed to AQP1. We have reconstituted purified protein from bovine and human red blood cell membranes into highly ordered 2D crystals. The topography of both AQP1s was determined by electron microscopy from freeze-dried, unidirectionally metal-shadowed 2D crystals as well as from surface topographs of native crystals recorded in buffer solution with the atomic force microscope (AFM). In spite of the high level of sequence homology between bovine and human AQP1, the surfaces showed distinct differences. Alignment of both sequences and comparison of the acquired surface topographies with the atomic model of human AQP1 revealed the topographic changes on the surface of bovine AQP1 to be induced by a few amino acid substitutions. A striking degree of sequence homology was found between the carboxyl-terminal domains of AQP1s from different organisms and EF-hands from Ca2+-binding proteins belonging to the calmodulin superfamily, suggesting the existence of a Ca2+-binding site at the C terminus of AQP1 instead of the putative cGMP-binding site reported previously. To unveil its position on the acquired surface topographies, 2D crystals of AQP1 were digested with carboxypeptidase Y, which cleaves off the intracellular C terminus. Difference maps of AFM topographs between the native and the peptidase-treated AQP1s showed the carboxylic tail to be close to the 4-fold symmetry axis of the tetramer. SDS-PAGE and matrix-assisted laser desorption/ionisation mass spectrometry of native and decarboxylated bovine and human AQP1 revealed that the EF-hand motif found at the C terminus of AQP1 was partially resistant to peptidase digestion. The importance of the C-terminal domain is implicated by structural instability of decarboxylated AQP1. A possible role of the C terminus and calcium in translocation of AQP1 in cholangiocytes from intracellular vesicles to the plasma membrane and in triggering its fusion is discussed. Functional studies are now required to identify the physiological role of the Ca2+-binding site.     

Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

Aquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca2+-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg2+-dependent, Ca2+-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1.     

Influence of the Cytoplasmic Domains of Aquaporin-4 on Water Conduction and Array Formation

Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 Å resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 Å resolution. The features of the 10-Å density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-Å map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform.     

Structure and stability of the spinach aquaporin SoPIP2;1 in detergent micelles and lipid membranes

Background

SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1.

Methodology/Principal Finding

We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-β-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly α-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58°C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70°C.

Conclusion/Significance

The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications.     

The 4.5 A structure of human AQP2

Located in the principal cells of the collecting duct, aquaporin-2 (AQP2) is responsible for the regulated water reabsorption in the kidney and is indispensable for the maintenance of body water balance. Disregulation or malfunctioning of AQP2 can lead to severe diseases such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. Here we present the crystallization of recombinantly expressed human AQP2 into two-dimensional protein-lipid arrays and their structural characterization by atomic force microscopy and electron crystallography. These crystals are double-layered sheets that have a diameter of up to 30 microm, diffract to 3 A(-1) and are stacked by contacts between their cytosolic surfaces. The structure determined to 4.5 A resolution in the plane of the membrane reveals the typical aquaporin fold but also a particular structure between the stacked layers that is likely to be related to the cytosolic N and C termini.     

Crystal structure of a Y35G mutant of bovine pancreatic trypsin inhibitor.

The structure of a Y35G mutant of bovine pancreatic trypsin inhibitor (BPTI) was solved by molecular replacement and was refined by both simulated annealing and restrained least-squares at 1.8 A resolution. The crystals belong to the space group P42212, with unit cell dimensions a = b = 46.75 A, c = 50.61 A. The final R-factor is 0.159 and the deviation from ideality for bond distances is 0.02 A. The structure of the mutant differs from that of the native protein, showing an overall root-mean-square (r.m.s.) difference of 1.86 A for main-chain atoms. However, the change is mostly localized in the two loops (respective r.m.s. values of 2.04 A and 3.93 A) and the C terminus (r.m.s. 6.79 A), while the core of the protein is well conserved (r.m.s. 0.45 A). The change in the loop regions can be clearly attributed to the mutation while the difference in the C terminus might be only due to a different crystal packing. Seventy water molecules were included in the model but only seven of them are shared with the native structure. Thermal parameters are showing a good correlation with those for the wild-type of BPTI.     

Crystal structure of the complementary quadruplex formed by d(GCATGCT) at atomic resolution

Here we report the crystal structure of the DNA heptanucleotide sequence d(GCATGCT) determined to a resolution of 1.1 Å. The sequence folds into a complementary loop structure generating several unusual base pairings and is stabilised through cobalt hexammine and highly defined water sites. The single stranded loop is bound together through the G(N2)–C(O2) intra-strand H-bonds for the available G/C residues, which form further Watson–Crick pairings to a complementary sequence, through 2-fold symmetry, generating a pair of non-planar quadruplexes at the heart of the structure. Further, four adenine residues stack in pairs at one end, H-bonding through their N7–N6 positions, and are additionally stabilised through two highly conserved water positions at the structural terminus. This conformation is achieved through the rotation of the central thymine base at the pinnacle of the loop structure, where it stacks with an adjacent thymine residue within the lattice. The crystal packing yields two halved biological units, each related across a 2-fold symmetry axis spanning a cobalt hexammine residue between them, which stabilises the quadruplex structure through H-bonds to the phosphate oxygens and localised hydration.     

Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X-ray diffraction to 1.35 A resolution and by neutron diffraction to 2.1 A resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X-ray diffraction, which were verified by comparison to D2O-H2O difference neutron scattering density. Six of 184 water molecules in the X-ray structure, all with B-factors greater than 50 A2, were found to be spurious after comparison with neutron results. Roughly two-thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one-half of the water of hydration was located by X-ray diffraction. Polar regions of the protein are well hydrated, and significant D2O-H2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures. Solvent models found in seven other bovine trypsin and trypsinogen and rat trypsin structures determined by X-ray diffraction were compared. Internal water molecules are well conserved in all trypsin structures including anionic rat trypsin, which is 65% homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were also found in trypsinogen, suggesting that they are located in regions of the apoprotein that are structurally conserved in the transition to the mature protein. Seven waters were displaced upon activation of trypsinogen. Water structure at crystal contacts is not generally conserved in different crystal forms. Three groups of integral structural water molecules are highly conserved in all solvent structures, including a spline of water molecules inserted between two beta-strands, which may resemble an intermediate in the formation of beta sheets during the folding of a protein.     

Structure of a T4 hairpin loop on a Z-DNA stem and comparison with A-RNA and B-DNA loops

The synthetic DNA oligomer C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G crystallizes as a Z-DNA hexamer, capped at one end by a T4 loop. The crystals are monoclinic, space group C2, with a = 57.18 A, b = 21.63 A, c = 36.40 A, beta = 95.22 degrees, and one hairpin molecule per asymmetric unit. The structure of the z-hexamer stem was determined by molecular replacement, and the T4 loop was positioned by difference map methods. The final R factor at 2.1 A resolution for hairpin plus 70 water molecules is 20% for 2 sigma data, with a root-mean-square error of 0.26 A. The (C-G)3 stem resembles the free Z-DNA hexamer with minor crystal packing effects. The T4 loop differs from that observed on a B-DNA stem in solution, or in longer loops in tRNA, in that it shows intraloop and intermolecular interactions rather than base stacking on the final base-pair of the stem. Bases T7, T8 and T9 stack with one another and with the sugar of T7. Two T10 bases from different molecules stack between the C1-G12 terminal base-pairs of a third and fourth molecule, to simulate a T.T "base-pair". Distances between thymine N and O atoms suggest that the two thymine bases are hydrogen bonded, and a keto-enol tautomer pair is favored over disordered keto-keto wobble pairs. The hairpin molecules pack in the crystal in herringbone columns in a manner that accounts well for the observed relative crystal growth rates in a, b and c directions. Hydration seems to be most extensive around the phosphate groups, with lesser hydration within the grooves.     

Two-dimensional crystallization of Escherichia coli lactose permease

A chimeric protein consisting of lactose permease with cytochrome b562 in the middle cytoplasmic loop and six His residues at the C terminus (LacY/L6cytb562/417H6 or "red permease") was overexpressed in Escherichia coli and isolated by nickel affinity chromatography after solubilization with dodecyl-beta,d-maltopyranoside. Red permease was then reconstituted in the presence of phospholipids, yielding densely packed vesicles and well-ordered two-dimensional (2D) crystals as shown by electron microscopy of negatively stained specimens. Single-particle analysis of 16 383 protein particles in densely packed vesicles reveals a 5.4-nm-long trapeziform protein of 4.1 to 5.1 nm width, with a central stain-filled indentation. Depending on reconstitution conditions, trigonal and rectangular crystallographic packing arrangements of these elongated particles assembled into trimers are observed. The best ordered 2D crystals exhibit a rectangular unit cell, of dimensions a = 9.9 nm, b = 17.4 nm, that houses two trimeric complexes. Projection maps calculated to a resolution of 2 nm show that these crystals consist of two layers.     

Specific plasma membrane aquaporins of the PIP1 subfamily are expressed in sieve elements and guard cells

BACKGROUND INFORMATION: Transmembrane water flow is aided by water-specific channel proteins, aquaporins. Plant genomes code for approx. 35 expressed and functional aquaporin isoforms. Plant aquaporins fall into four different subfamilies of which the PIPs (plasma membrane intrinsic proteins) constitute the largest and evolutionarily most conserved subfamily with 13 members in Arabidopsis and maize. Furthermore, the PIPs can be divided into two phylogenetic groups, PIP1 and PIP2, of which the PIP1 isoforms are most tightly conserved, sharing >90% amino acid sequence identity. As the nomenclature implies, the majority of PIPs have been shown to be localized at the plasma membrane. Recently, two highly abundant plasma membrane aquaporins, SoPIP2;1 and SoPIP1;2, have been purified and structurally characterized. RESULTS: We report the cloning of a cDNA encoding SoPIP1;2 and show that there are at least five additional sequences homologous with SoPIP2;1 and SoPIP1;2 in the spinach genome. To understand their role in planta, we have investigated the cellular localization of the aquaporin homologues SoPIP1;2 and SoPIP1;1. By Western- and Northern-blot analyses and by immunocytochemical detection at the light and electron microscopic levels, we show that SoPIP1;2 is highly expressed in phloem sieve elements of leaves, roots and petioles and that SoPIP1;1 is present in stomatal guard cells. CONCLUSIONS: Localization of the two abundant aquaporin isoforms suggests roles for specific PIPs of the PIP1 subgroup in phloem loading, transport and unloading, and in stomatal movements.     

Solution structure of the VEGF-binding domain of Flt-1: comparison of its free and bound states.

The extracellular portion of the VEGF and PlGF receptor, Flt-1 (or VEGFR-1), consists of seven immunoglobulin-like domains. The second domain from the N terminus (Flt-1D2) is necessary and sufficient for high affinity VEGF binding. The 1.7 A resolution crystal structure of Flt-1D2 bound to VEGF revealed that this domain is a member of the I-set of the immunoglobulin superfamily, but has several unusual features including a region near the N terminus that bulges away from the domain rather than pairing with the neighboring beta-strand. Some of the residues in this region make contact with VEGF, raising the possibility that this bulge could be a consequence of VEGF binding and might not be present in the absence of ligand. Here we report the three-dimensional structure of Flt-1D2 in its uncomplexed form determined by NMR spectroscopy. A semi-automated method for NOE assignment that takes advantage of the previously solved crystal structure was used to facilitate rapid analysis of the 3D NOESY spectra. The solution structure is very similar to the previously reported VEGF-bound crystal structure; the N-terminal bulge is present, albeit in a different conformation. We also report the 2.7 A crystal structure of Flt-1D2 in complex with VEGF solved in a different crystal form that reveals yet another conformation for the N-terminal bulge region. (1)H-(15)N heteronuclear NOEs indicate this region is flexible in solution; the crystal structures show that this region is able to adopt more than one conformation even when bound to VEGF. Thus, VEGF-binding is not accompanied by significant structural change in Flt-1D2, and the unusual structural features of Flt-1D2 are an intrinsic property of this domain.     

The beta-strand D of transthyretin trapped in two discrete conformations

Conformational changes in native and variant forms of the human plasma protein transthyretin (TTR) induce several types of amyloid diseases. Biochemical and structural studies have mapped the initiation site of amyloid formation onto residues at the outer C and D beta-strands and their connecting loop. In this study, we characterise an engineered variant of transthyretin, Ala108Tyr/Leu110Glu, which is kinetically and thermodynamically more stable than wild-type transthyretin, and as a consequence less amyloidogenic. Crystal structures of the mutant were determined in two space groups, P2(1)2(1)2 and C2, from crystals grown in the same crystallisation set-up. The structures are identical with the exception for residues Leu55-Leu58, situated at beta-strand D and the following DE loop. In particular, residues Leu55-His56 display large shifts in the C2 structure. There the direct hydrogen bonding between beta-strands D and A has been disrupted and is absent, whereas the beta-strand D is present in the P2(1)2(1)2 structure. This difference shows that from a mixture of metastable TTR molecules, only the molecules with an intact beta-strand D are selected for crystal growth in space group P2(1)2(1)2. The packing of TTR molecules in the C2 crystal form and in the previously determined amyloid TTR (ATTR) Leu55Pro crystal structure is close-to-identical. This packing arrangement is therefore not unique in amyloidogenic mutants of TTR.     

The crystal structure of the Epstein-Barr virus protease shows rearrangement of the processed C terminus

Epstein-Barr virus (EBV) belongs to the gamma-herpesvirinae subfamily of the Herpesviridae. The protease domain of the assemblin protein of herpesviruses forms a monomer-dimer equilibrium in solution. The protease domain of EBV was expressed in Escherichia coli and its structure was solved by X-ray crystallography to 2.3A resolution after inhibition with diisopropyl-fluorophosphate (DFP). The overall structure confirms the conservation of the homodimer and its structure throughout the alpha, beta, and gamma-herpesvirinae. The substrate recognition could be modelled using information from the DFP binding, from a crystal contact, suggesting that the substrate forms an antiparallel beta-strand extending strand beta5, and from the comparison with the structure of a peptidomimetic inhibitor bound to cytomegalovirus protease. The long insert between beta-strands 1 and 2, which was disordered in the KSHV protease structure, was found to be ordered in the EBV protease and shows the same conformation as observed for proteases in the alpha and beta-herpesvirus families. In contrast to previous structures, the long loop located between beta-strands 5 and 6 is partially ordered, probably due to DFP inhibition and a crystal contact. It also contributes to substrate recognition. The protease shows a specific recognition of its own C terminus in a binding pocket involving residue Phe210 of the other monomer interacting across the dimer interface. This suggests conformational changes of the protease domain after its release from the assemblin precursor followed by burial of the new C terminus and a possible effect onto the monomer-dimer equilibrium. The importance of the processed C terminus was confirmed using a mutant protease carrying a C-terminal extension and a mutated release site, which shows different solution properties and a strongly reduced enzymatic activity.     

Membrane skeletal association and post‐translational allosteric regulation of Toxoplasma gondii GAPDH1

When Toxoplasma gondii egresses from the host cell, glyceraldehyde‐3‐phosphate dehydrogenase 1 (GAPDH1), which is primary a glycolysis enzyme but actually a quintessential multifunctional protein, translocates to the unique cortical membrane skeleton. Here, we report the 2.25 Å resolution crystal structure of the GAPDH1 holoenzyme in a quaternary complex providing the basis for the molecular dissection of GAPDH1 structure–function relationships Knockdown of GAPDH1 expression and catalytic site disruption validate the essentiality of GAPDH1 in intracellular replication but we confirmed that glycolysis is not strictly essential. We identify, for the first time, S‐loop phosphorylation as a novel, critical regulator of enzymatic activity that is consistent with the notion that the S‐loop is critical for cofactor binding, allosteric activation and oligomerization. We show that neither enzymatic activity nor phosphorylation state correlate with the ability to translocate to the cortex. However, we demonstrate that association of GAPDH1 with the cortex is mediated by the N‐terminus, likely palmitoylation. Overall, glycolysis and cortical translocation are functionally decoupled by post‐translational modifications.