Novel arrangement of immunoglobulin variable domains: X-ray crystallographic analysis of the lambda-chain dimer Bence-Jones protein Loc

We have characterized and crystallized a human lambda I light-chain dimer, Bence-Jones protein Loc, which has variable (V) region antigenic determinants characteristic for the lambda I subgroup and constant (C) region determinants of the C lambda I gene Mcg. The crystal structure was determined to 3-A resolution; the R factor is 0.27. The angle formed by the twofold axes of the V and C domains, the "elbow bend", is 97 degrees, the smallest found so far for an antibody fragment. The antigen-binding site formed by the two V domains of the Loc light chain differs significantly from those of other immunoglobulin molecules (light-chain dimers and Fab fragments) for which X-ray crystallographic data are available. Whereas, in other antibody fragments, the V domains are related by a local twofold axis, a local twofold screw axis with a translational component of 3.5 A relates the V domains in protein Loc. In contrast to the classic antigen binding "pocket" formed by V domain interactions in the previously characterized antibody structures, the V region associations in protein Loc result in a central protrusion in the binding site, with grooves on two sides of the protrusion. The structure of protein Loc indicates that immunoglobulins are physically capable of forming a more diverse spectrum of antigen-binding sites than has been heretofore apparent. Moreover, the unusual protruding nature of the binding site may be analogous to structures required for some anti-idiotypic antibodies. Further, the complementarity-determining residues form parts of two independent grooves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of a second crystal form of Bence-Jones protein Loc: strikingly different domain associations in two crystal forms of a single protein

We have determined the structure of the immunoglobulin light-chain dimer Loc in a second crystal form that was grown from distilled water. The crystal structure was determined to 2.8-A resolution; the R factor is 0.22. The two variable domains are related by local 2-fold axes and form an antigen binding "pocket". The variable domain-variable domain interaction observed in this crystal form differs from the one exhibited by the protein when crystallized from ammonium sulfate in which the two variable domains formed a protrusion (Chang et al., 1985). The structure attained in the distilled water crystals is similar to, but not identical with, the one observed for the Mcg light-chain dimer in crystals grown from ammonium sulfate. Thus, two strikingly different structures were attained by this multisubunit protein in crystals grown under two different, commonly used, crystallization techniques. The quaternary interactions exhibited by the protein in the two crystal forms are sufficiently different to suggest fundamentally different interpretations of the structural basis for the function of this protein. This observation may have general implications regarding the use of single crystallographic determinations for detailed identification of structural and functional relationships. On the other hand, proteins whose structures can be altered by manipulation of crystallization conditions may provide useful systems for study of fundamental structural chemistry.     

Crystal structure of a cyclomaltoheptaose-4-hydroxybiphenyl inclusion complex

The crystal structure of the inclusion complex of cyclomaltoheptaose (beta-cyclodextrin) with 4-hydroxybiphenyl was determined by single-crystal X-ray diffraction at 150K. The complex contains two cyclomaltoheptaose molecules, two 4-hydroxybiphenyl molecules, one ethanol molecule and fifteen water molecules in the asymmetric unit, and could be formulated as [2(C(42)H(70)O(35)).2(C(12)H(10)O).(C(2)H(6)O).15(H(2)O)]. It crystallized in the triclinic space group P1 with unit cell constants a=15.257(3), b=15.564(3), c=15.592(2)A, alpha=104.485(15) degrees , beta=101.066(14) degrees , gamma=104.330(17) degrees , V=3,343.6(10)A(3). In the crystal lattice, two beta-cyclodextrins form a head-to-head dimer jointed through hydrogen bonds. Two 4-hydroxybiphenyls were included in the dimer cavity with their hydroxyl groups protruding from two primary hydroxyl sides of the cyclodextrin molecules. The guest 4-hydroxybiphenyl molecules linked into a chain via a combination of an O-Hcdots, three dots, centeredO hydrogen bond and face-to-face pi-pi stacking of the phenyl rings. The crystal structure supports the calculation results indicating that the 2:2 inclusion complex formed by beta-cyclodextrin and 4-hydroxybiphenyl is the energetically favored structure.     

Structure of a four-way bridged ParB-DNA complex provides insight into P1 segrosome assembly

The plasmid partition process is essential for plasmid propagation and is mediated by par systems, consisting of centromere-like sites and two proteins, ParA and ParB. In the first step of partition by the archetypical P1 system, ParB binds a complicated centromere-like site to form a large nucleoprotein segrosome. ParB is a dimeric DNA-binding protein that can bridge between both A-boxes and B-boxes located on the centromere. Its helix-turn-helix domains bind A-boxes and the dimer domain binds B-boxes. Binding of the first ParB dimer nucleates the remaining ParB molecules onto the centromere site, which somehow leads to the formation of a condensed segrosome superstructure. To further understand this unique DNA spreading capability of ParB, we crystallized and determined the structure of a 1:2 ParB-(142-333):A3-B2-box complex to 3.35A resolution. The structure reveals a remarkable four-way, protein-DNA bridged complex in which both ParB helix-turn-helix domains simultaneously bind adjacent A-boxes and the dimer domain bridges between two B-boxes. The multibridging capability and the novel dimer domain-B-box interaction, which juxtaposes the DNA sites close in space, suggests a mechanism for the formation of the wrapped solenoid-like segrosome superstructure. This multibridging capability of ParB is likely critical in its partition complex formation and pairing functions.     

Crystallization and preliminary X-ray crystallographic analysis of Ace: a collagen-binding MSCRAMM from Enterococcus faecalis

Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3(1)21 or P3(2)21 with unit cell parameters a=b=80.24, c=105.91 A; alpha=beta=90 and gamma=120 degrees. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.     

Cloning,expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor.

T-lymphocytes recognize a wide variety of antigens through highly diverse cell-surface glycoproteins known as T-cell receptors (TCRs). These disulfide-linked heterodimers are composed of alpha and beta or gamma and delta polypeptide chains consisting of variable (V) and constant (C) domains non-covalently associated with at least four invariant chains to form the TCR-CD3 complex. It is well established that alpha beta TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three-dimensional structure of alpha beta TCRs has recently become available through X-ray crystallography. In contrast, the antigen specificity of gamma delta TCRs is much less well understood and their three-dimensional structure is unknown. We have cloned the delta chain of a human TCR specific for the MHC class I HLA-A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli. Following affinity purification using a nickel chelate adsorbent, the recombinant V delta domain was crystallized in a form suitable for X-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 A. and diffract to beyond 2.3 A resolution. The ability of a V delta domain produced in bacteria to form well-ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of gamma delta TCRs.     

Crystal structure of the V domain of human Nectin-like molecule-1/Syncam3/Tsll1/Igsf4b, a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule

Nectins are Ca(2+)-independent immunoglobulin (Ig) superfamily proteins that participate in the organization of epithelial and endothelial junctions. Nectins have three Ig-like domains in the extracellular region, and the first one is essential in cell-cell adhesion and plays a central role in the interaction with the envelope glycoprotein D of several viruses. Five Nectin-like molecules (Necl-1 through -5) with similar domain structures to those of Nectins have been identified. Necl-1 is specifically expressed in neural tissue, has Ca(2+)-independent homophilic and heterophilic cell-cell adhesion activity, and plays an important role in the formation of synapses, axon bundles, and myelinated axons. Here we report the first crystal structure of its N-terminal Ig-like V domain at 2.4 A, providing insight into trans-cellular recognition mediated by Necl-1. The protein crystallized as a dimer, and the dimeric form was confirmed by size-exclusion chromatography and chemical cross-linking experiments, indicating this V domain is sufficient for homophilic interaction. Mutagenesis work demonstrated that Phe(82) is a key residue for the adhesion activity of Necl-1. A model for homophilic adhesion of Necl-1 at synapses is proposed based on its structure and previous studies.     

Structure of a hinge-bending bacteriophage T4 lysozyme mutant, Ile3-->Pro.

The mutant T4 phage lysozyme in which isoleucine 3 is replaced by proline (I3P) crystallizes in an orthorhombic form with two independent molecules in the asymmetric unit. Relative to wild-type lysozyme, which crystallizes in a trigonal form, the two I3P molecules undergo large hinge-bending displacements with the alignments of the amino-terminal and carboxy-terminal domains changed by 28.9 degrees and 32.9 degrees, respectively. The introduction of the mutation, together with the hinge-bending displacement, is associated with repacking of the side-chains of Phe4, Phe67 and Phe104. These aromatic residues are clustered close to the site of the mutation and are at the junction between the amino and carboxyl-terminal domains. As a result of this structural rearrangement the side-chain of Phe4 moves from a relatively solvent-exposed conformation to one that is largely buried. Mutant I3P also crystallizes in the same trigonal form as wild-type and, in this case, the observed structural changes are restricted to the immediate vicinity of the replacement. The main change is a shift of 0.3 to 0.5 A in the backbone of residues 1 to 5. The ability to crystallize I3P under similar conditions but in substantially different conformations suggests that the molecule undergoes large-scale hinge-bending displacements in solution. It is also likely that these conformational excursions are associated with repacking at the junction of the N-terminal and C-terminal domains. On the other hand, the analysis is complicated by possible effects of crystal packing. The different I3P crystal structures show substantial differences in the binding of solvent, both at the site of the Ile3-->Pro replacement and at other internal sites.     

The N-terminal domain of betaB2-crystallin resembles the putative ancestral homodimer

betagamma-crystallins from the eye lens are proteins consisting of two similar domains joined by a short linker. All three-dimensional structures of native proteins solved so far reveal similar pseudo-2-fold pairing of the domains reflecting their presumed ancient origin from a single-domain homodimer. However, studies of engineered single domains of members of the betagamma-crystallin superfamily have not revealed a prototype ancestral solution homodimer. Here we report the 2.35 A X-ray structure of the homodimer of the N-terminal domain of rat betaB2-crystallin (betaB2-N). The two identical domains pair in a symmetrical manner very similar to that observed in native betagamma-crystallins, where N and C-terminal domains (which share approximately 35% sequence identity) are related by a pseudo-2-fold axis. betaB2-N thus resembles the ancestral prototype of the betagamma-crystallin superfamily as it self-associates in solution to form a dimer with an essentially identical domain interface as that between the N and C domains in betagamma-crystallins, but without the benefit of a covalent linker. The structure provides further evidence for the role of two-domain pairing in stabilising the protomer fold. These results support the view that the betagamma-crystallin superfamily has evolved by a series of gene duplication and fusion events from a single-domain ancestor capable of forming homodimers.     

Crystal structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVAII) hydrolyzing cyclodextrins and pullulan at 2.6 A resolution

The crystal structure of Thermoactinomyces vulgaris R-47 alpha-Amylase II (TVAII) has been determined by multiple isomorphous replacement at 2.6 A resolution. TVAII was crystallized in an orthorhombic system with the space group P212121 and the cell dimensions a=118.5 A, b=119.5 A, c=114.5 A. There are two molecules in an asymmetric unit, related by the non-crystallographic 2-fold symmetry. Diffraction data were collected at 113 K and the cell dimensions reduced to a=114.6 A, b=117.9 A, c=114.2 A, and the model was refined against 7.0-2.6 A resolution data giving an R-factor of 0.204 (Rfree=0.272). The final model consists of 1170 amino acid residues (two molecules) and 478 water molecules with good chemical geometry. TVAII has three domains, A, B, and C, like other alpha-amylases. Domain A with a (beta/alpha)8 barrel structure and domain C with a beta-sandwich structure are very similar to those found in other alpha-amylases. Additionally, TVAII has an extra domain N composed of 121 amino acid residues at the N-terminal site, which has a beta-barrel-like structure consisting of seven antiparallel beta-strands. Domain N is one of the driving forces in the formation of the dimer structure of TVAII, but its role in the enzyme activity is still not clear. TVAII does not have the Ca2+ binding site that connects domains A and B in other alpha-amylases, rather the NZ atom of Lys299 of TVAII serves as the connector between these domains. TVAII can hydrolyze cyclodextrins and pullulan as well as starch. Based on a structural comparison with the complex between a mutant cyclodextrin glucanotransferase and a beta-cyclodextrin derivative, Phe286 located at domain B is considered the residue most likely to recognize the hydrophobic cavity of cyclodextrins. The active-site cleft of TVAII is wider and shallower than that of other alpha-amylases, and seems to be suitable for the binding of pullulan which is expected not to adopt the helical structure of amylose.     

Conformational flexibility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: implications for lipid binding

An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.     

Crystal structure of the FHA domain of the Chfr mitotic checkpoint protein and its complex with tungstate

The Chfr mitotic checkpoint protein is frequently inactivated in human cancer. We determined the three-dimensional structure of its FHA domain in its native form and in complex with tungstate, an analog of phosphate. The structures revealed a beta sandwich fold similar to the previously determined folds of the Rad53 N- and C-terminal FHA domains, except that the Rad53 domains were monomeric, whereas the Chfr FHA domain crystallized as a segment-swapped dimer. The ability of the Chfr FHA domain to recognize tungstate suggests that it shares the ability with other FHA domains to bind phosphoproteins. Nevertheless, differences in the sequence and structure of the Chfr and Rad53 FHA domains suggest that FHA domains can be divided into families with distinct binding properties.     

Self base pairing in a complementary deoxydinucleoside monophosphate duplex: crystal and molecular structure of deoxycytidylyl-(3'-5')-deoxyguanosine

The molecular structure of ammonium deoxycytidylyl-(3'-5')-deoxyguanosine, crystallized from aqueous acetone near pH 4, was determined for X-ray diffraction data. The crystals were tetragonal, space group P43212 with a = b = 11.078 (1) A and c = 45.826 (4) A. The structure was solved by tangent expansion of phases based on a derived phosphorus position and refined to R = 0.060 by full matrix least squares. Molecules related by a 2-fold symmetry axis are connected by hydrogen bonds between the bases and form parallel right-handed duplexes. Pairs of cytosines share a proton at N(3) and are joined by three hydrogen bonds: N(4)-H...O(2)...H-N(4), and N(3)-H...N(3). Guanines are joined by two hydrogen bonds: N(2)-H...N(3) and N(3)...H-N(2). Base-stacking interactions within the duplex are weak with the cytosine and guanine ring planes inclined at 24 degrees to each other in each monomer. Despite the unusual arrangement of the molecules, the sugar phosphate backbone has the g-g- conformation normally associated with right-handed double helical structures. Conformational parameters of the nucleosides are also typical with both sugars C(2')-endo and glycosidic torsion angles 55 degrees for cytidine and 94 degrees for guanosine. The bonding geometry of the bases is influenced by hydrogen bonding and charge-transfer networks in the crystal lattice. The solvent molecules interact with the dimer in three fused circular hydrogen bonding domains with a single disordered ammonium cation per d(CpG) dimer. Parallels with the formation of self base pairs and their implications in molecular biology are discussed.     

Circular permutation of betaB2-crystallin changes the hierarchy of domain assembly.

The betagamma-crystallins form a superfamily of eye lens proteins comprised of multiple Greek motifs that are symmetrically organized into domains and higher assemblies. In the betaB2-crystallin dimer each polypeptide folds into two similar domains that are related to monomeric gamma-crystallin by domain swapping. The crystal structure of the circularly permuted two-domain betaB2 polypeptide shows that permutation converts intermolecular domain pairing into intramolecular pairing. However, the dimeric permuted protein is, in fact, half a native tetramer. This result shows how the sequential order of domains in multi-domain proteins can affect quaternary domain assembly.     

Intercalation of water molecules between nucleic acid bases in the crystal structures of 6-azathymine hemihydrate and 5-amino-2-thiocytosine dihydrochloride dihydrate

The crystal structure of 6-azathymine hemihydrate (6AzTH) exhibits a novel intercalation of water molecules interposed half-way between the modified bases 6.3 to 6.7 A apart. The crystal contains four molecules of 6-azathymine (6AzT) and two water molecules as the independent repeating unit. These two water molecules together with the four bases form two separate water sandwiches. In the crystal structure these sandwiches form two sets of local clusters. The anhydrous crystalline form of 6AzT, on the other hand, is stabilized by base stacking interactions. Both the water molecules in 6AzTH that are involved in sandwich formation have trigonal coordination around them. A reexamination of the crystal structure of 5-amino-2-thiocytosine (5A2TC) revealed that one of the water molecules in this structure also forms a water sandwich and has trigonal coordination whereas the other water molecule with tetrahedral coordination does not form a sandwich. The environment and the characteristics of the intercalated water molecule in these structures suggest a possible role for such water intercalations in the dynamics of DNA. Crystals of 6AzTH are monoclinic, space group P21/n, with unit cell parameters a = 8.861 (1), b = 13.177 (3), c = 20.662 (2) A, beta = 93.35 (1) degrees, and Z = 16. From diffractometer data (2503 reflections, greater than or equal to 3 sigma), the crystal structure was solved and refined to an R of 0.056.     

Hinge-loop mutation can be used to control 3D domain swapping and amyloidogenesis of human cystatin C

Cystatins are natural inhibitors of cysteine proteases, enzymes that are widely distributed in animals, plants, and microorganisms. Human cystatin C (hCC) has been also recognized as an aggregating protein directly involved in the formation of pathological amyloid fibrils, and these amyloidogenic properties greatly increase in a naturally occurring L68Q hCC variant. For a long time only dimeric structure of wild-type hCC has been known. The dimer is created through 3D domain swapping process, in which two parts of the cystatin structure become separated from each other and next exchanged between two molecules. Important role in the domain swapping plays the L1 loop, which connects the exchanging segments and, upon dimerization, transforms from a β-turn into a part of a long β-strand. In the very recently published first monomeric structure of human cystatin C (hCC-stab1), dimerization was abrogated due to clasping of the β-strands from the swapping domains by an engineered disulfide bridge. We have designed and constructed another mutated cystatin C with the smallest possible structural intervention, that is a single-point mutation replacing hydrophobic V57 from the L1 loop by polar asparagine, known as a stabilizer of a β-turn motif. V57N hCC mutant occurred to be stable in its monomeric form and crystallized as a monomer, revealing typical cystatin fold with a five-stranded antiparallel β-sheet wrapped around an α-helix. Here we report a 2.04 Å resolution crystal structure of V57N hCC and discuss the architecture of the protein in comparison to chicken cystatin, hCC-stab1 and dimeric hCC.     

Structure and dynamics of the second and third transmembrane domains of human glycine receptor

A 61-residue polypeptide resembling the second and third transmembrane domains (TM23) of the alpha-1 subunit of human glycine receptor and its truncated form, both with the wild-type loop linking the two TM domains (the "23" loop), were studied using high-resolution NMR. Well-defined domain structures can be identified for the TM2, 23 loop, and TM3 regions. Contrary to the popular model of a long and straight alpha-helical structure for the pore-lining TM2 domain for the Cys-loop receptor family, the last three residues of the TM2 domain and the first eight residues of the 23 loop (S16-S26) seem to be intrinsically nonhelical and highly flexible even in trifluoroethanol, a solvent known to promote and stabilize alpha-helical structures. The six remaining residues of the 23 loop and most of the TM3 domain exhibit helical structures with a kinked pi-helix (or a pi-turn) from W34 to C38 and a kink angle of 159 +/- 3 degrees . The tertiary fold of TM3 relative to TM2 is defined by several unambiguously identified long-range NOE cross-peaks within the loop region and between TM2 and TM3 domains. The 20 lowest-energy structures show a left-handed tilt of TM3 relative to TM2 with a tilting angle of 44 +/- 2 degrees between TM2 (V1-Q14) and TM3 (L39-E48) helix axes. This left-handed TM2-TM3 arrangement ensures a neatly packed right-handed quaternary structure of five subunits to form an ion-conducting pore. This is the first time that two TM domains of the glycine receptor linked by the important 23 loop have ever been analyzed at atomistic resolution. Many structural characteristics of the receptor can be inferred from the structural and dynamical features identified in this study.     

What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR

Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the peptidoglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high- resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (approximately 17 degrees closure from solution to X-ray structures). A comparison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant. Dipolar couplings were also measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). In this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (approximately 11 degrees). Direct refinement of lysozyme crystal structures with the measured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond. The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure.     

Structure of recombinant Haemophilus influenzae e (P4) acid phosphatase reveals a new member of the haloacid dehalogenase superfamily

Lipoprotein e (P4) from Haemophilus influenzae belongs to the "DDDD" superfamily of phosphohydrolases and is the prototype of class C nonspecific acid phosphatases. P4 is also a component of a H. influenzae vaccine. We report the crystal structures of recombinant P4 in the ligand-free and tungstate-inhibited forms, which are the first structures of a class C phosphatase. P4 has a two-domain architecture consisting of a core alpha/beta domain and a smaller alpha domain. The core domain features a five-stranded beta-sheet flanked by helices on both sides that is reminiscent of the haloacid dehalogenase superfamily. The alpha domain appears to be unique and plays roles in substrate binding and dimerization. The active site is solvent accessible and located in a cleft between the two domains. The structure shows that P4 is a metalloenzyme and that magnesium is the most likely metal ion in the crystalline recombinant enzyme. The ligands of the metal ion are the carboxyl groups of the first and third Asp residues of the DDDD motif, the backbone carbonyl of the second Asp of the DDDD motif, and two water molecules. The structure of the tungstate-bound enzyme suggests that Asp64 is the nucleophile that attacks the substrate P atom. Dimerization appears to be important for catalysis because intersubunit contacts stabilize the active site. Analysis of the structural context of mutations engineered for vaccine studies shows that the most promising mutations are located in the dimer interface. This observation suggests a structure-based vaccine design strategy in which the dimer interface is disrupted in order to expose epitopes that are buried in dimeric P4.     

Molecular packing and intermolecular interactions in two structural polymorphs of N-palmitoylethanolamine, a type 2 cannabinoid receptor agonist

The molecular structure, packing properties, and intermolecular interactions of two structural polymorphs of N-palmitoylethanolamine (NPEA) have been determined by single-crystal X-ray diffraction. Polymorphs alpha and beta crystallized in monoclinic space group P2(1)/c and orthorhombic space group Pbca, respectively. In both polymorphs, NPEA molecules are organized in a tail-to-tail manner, resembling a bilayer membrane. Although the molecular packing in polymorph alpha is similar to that in N-myristoylethanolamine and N-stearoylethanolamine, polymorph beta is a new form. The acyl chains in both polymorphs are tilted by approximately 35 degrees with respect to the bilayer normal, with their hydrocarbon moieties packed in an orthorhombic subcell. In both structures, the hydroxy group of NPEA forms two hydrogen bonds with the hydroxy groups of molecules in the opposite leaflet, resulting in extended, zig-zag type H-bonded networks along the b-axis in polymorph alpha and along the a-axis in polymorph beta. Additionally, the amide N-H and carbonyl groups of adjacent molecules are involved in N-H...O hydrogen bonds that connect adjacent molecules along the b-axis and a-axis, respectively, in alpha and beta. Whereas in polymorph alpha the L-shaped NPEA molecules in opposite layers are arranged to yield a Z-like organization, in polymorph beta one of the two NPEA molecules is rotated 180 degrees , leading to a W-like arrangement. Lattice energy calculations indicate that polymorph alpha is more stable than polymorph beta by approximately 2.65 kcal/mol.