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The myelin sheath is a multilayered membrane that surrounds and insulates axons in the nervous system. One of the proteins specific to the peripheral nerve myelin is P2, a protein that is able to stack lipid bilayers. With the goal of obtaining detailed information on the structure–function relationship of P2, 14 structure-based mutated variants of human P2 were generated and produced. The mutants were designed to potentially affect the binding of lipid bilayers by P2. All mutated variants were also crystallized and preliminary crystallographic data are presented. The structural data from the mutants will be combined with diverse functional assays in order to elucidate the fine details of P2 function at the molecular level.

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a quantitatively major enzyme in myelin, where it localizes to the non-compact regions and is bound to the membrane surface. Although its catalytic activity in vitro has been characterized, the physiological function and in vivo substrate of CNPase remain unknown. Especially the N-terminal domain has been poorly characterized; previously, we have shown it is involved in CNPase dimerization and RNA binding. Here, we show that purified CNPase binds to the calcium sensor protein calmodulin (CaM) in a calcium-dependent manner; the binding site is in the N-terminal domain of CNPase. CaM does not affect the phosphodiesterase activity of CNPase in vitro, nor does it influence polyadenylic acid binding. The colocalization of CNPase and CaM during Schwann cell myelination in culture was observed, and CaM antagonists induced the colocalization of CNPase with microtubules in differentiated CG-4 oligodendrocytes. An analysis of post-translational modifications of CNPase from rat brain revealed the presence of two novel phosphorylation sites on Tyr110 and Ser169 within the N-terminal domain. The results indicate a role for the N-terminal domain of CNPase in mediating multiple molecular interactions and provide a starting point for detailed structure-function studies on CNPase and its N-terminal domain.

Information on very high pressure (VHP) effects on proteins is limited and therefore effects of VHP on chemistry, structure and function of two model proteins in medical use were studied. VHP (8 GPa) application to l-asparaginase (L-ASNase) resulted in faster mobility on clear native gels. VHP induced generation of lower-MW forms of L-ASNase but VHP treatment did not deteriorate asparaginase activity. Electrophoretic patterns in native and denaturing gels were comparable for untreated and pressurized recombinant human growth hormone (rhGH). rhGH function, however, was deteriorated as shown by a bioassay. In L-ASNase and rhGH a series of protein modifications and amino acid exchanges (indicating cleavage of covalent bonds) were revealed that may probably lead to functional and conformational changes. The findings have implications in protein chemistry, structure, and function and are useful for designing biotechnological applications of protein products.

A quantitatively major protein has been purified from the latex of Morus indica. The purified previously uncharacterized protein, M. indica lectin (MIL), was further shown to be a glycosylated tetramer and belongs to the family of jacalin-related lectins. Crystallization of MIL was also accomplished and the tetragonal crystals diffracted synchrotron X-rays to a resolution of 2.8 Å.

The death-associated protein kinase (DAPK) family has been characterized as a group of pro-apoptotic serine/threonine kinases that share specific structural features in their catalytic kinase domain. Two of the DAPK family members, DAPK1 and DAPK2, are calmodulin-dependent protein kinases that are regulated by oligomerization, calmodulin binding, and autophosphorylation. In this study, we have determined the crystal and solution structures of murine DAPK2 in the presence of the autoinhibitory domain, with and without bound nucleotides in the active site. The crystal structure shows dimers of DAPK2 in a conformation that is not permissible for protein substrate binding. Two different conformations were seen in the active site upon the introduction of nucleotide ligands. The monomeric and dimeric forms of DAPK2 were further analyzed for solution structure, and the results indicate that the dimers of DAPK2 are indeed formed through the association of two apposed catalytic domains, as seen in the crystal structure. The structures can be further used to build a model for DAPK2 autophosphorylation and to compare with closely related kinases, of which especially DAPK1 is an actively studied drug target. Our structures also provide a model for both homodimerization and heterodimerization of the catalytic domain between members of the DAPK family. The fingerprint of the DAPK family, the basic loop, plays a central role in the dimerization of the kinase domain.

Myelin is a tightly packed membrane multilayer in the nervous system, which harbours a specific set of quantitatively major proteins. All these proteins interact with the lipid bilayer, being either peripheral or integral membrane proteins. In this study, we examined the conformational properties of peptides from the myelin proteins P0, CNPase, MOBP, P2 and MOG, using trifluoroethanol and micelles of different detergents as membrane-like mimics. The peptides showed significant differences in their folding under the employed conditions, as evidenced by synchrotron radiation circular dichroism spectroscopy. Our experiments provide new structural information on the interactions between myelin proteins and membranes, using a simplified model system of synthetic peptides and micelles.

The myelin sheath is a crucial structure for the proper functioning of the vertebrate nervous system. We employ diverse methods to study the structure, function, and dynamics of the molecules specifically present in myelin. Eventually, we hope to better understand the details of the tightly packed myelin structure and the etiology of myelin-related diseases. The paper will provide background into the molecular structure of myelin, and recent results from our laboratory, dealing with the structure and function of selected myelin proteins, will be highlighted.

Death-associated protein kinase (DAPK) provides a model for calcium-bound calmodulin (CaM)–dependent protein kinases (CaMKs). Here, we report the crystal structure of the binary DAPK-CaM complex, using a construct that includes the DAPK catalytic domain and adjacent autoregulatory domain. When DAPK was in a complex with CaM, the DAPK autoregulatory domain formed a long seven-turn helix. This DAPK-CaM module interacted with the DAPK catalytic domain through two separate domain-domain interfaces, which involved the upper and the lower lobe of the catalytic domain. When bound to DAPK, CaM adopted an extended conformation, which was different from that in CaM-CaMK peptide complexes. Complementary biochemical analysis showed that the ability of DAPK to bind CaM correlated with its catalytic activity. Because many features of CaM binding are conserved in other CaMKs, our findings likely provide a generally applicable model for regulation of CaMK activity.

Background 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein consists of two domains: an uncharacterized N-terminal domain with little homology to other proteins, and a C-terminal phosphodiesterase domain. Findings In order to be able to fully characterize CNPase structurally and functionally, we have set up expression systems for different domains of CNPase, using a total of 18 different expression constructs. CNPase was expressed in E. coli with a TEV-cleavable His-tag. Enzymatic activity assays indicated that the purified proteins were active and correctly folded. The folding of both the full-length protein, as well as the N- and C-terminal domains, was also studied by synchrotron CD spectroscopy. A thermal shift assay was used to optimize buffer compositions to be used during purification and storage. The assay also indicated that CNPase was most stable at a pH of 5.5, and could be significantly stabilized by high salt concentrations. Conclusions We have been able to express and purify recombinantly several different domains of CNPase, including the isolated N-terminal domain, which is folded mainly into a β-sheet structure. The expression system can be used as an efficient tool to elucidate the role of CNPase in the myelin sheath.

Often, the amount of RNA that can be isolated from a defined tissue is very small. A method consisting of nondenaturing polyacrylamide gel electrophoresis and silver staining is described that can be used to evaluate the concentration of very dilute RNA samples. The method is a good starting point for assays dealing with small amounts of RNA, such as semiquantitative reverse transcription polymerase chain reaction (RT-PCR), making it possible to perform parallel assays from similar amounts of total RNA when quantitation by other methods is too insensitive. The method has been used successfully to monitor the amount of total RNA isolated from rat sciatic nerve and the rat C6 glioma cell line.

Gap junctions are formed by a family of transmembrane proteins, connexins. Connexin43 is a widely studied member of the family, being ubiquitously expressed in a variety of tissues and a target of a large number of disease mutations. The intracellular loop of connexin43 has been shown to include a calmodulin binding domain, but detailed 3-dimensional data on the structure of the complex are not available. In this study, we used a synthetic peptide from this domain to reveal the conformation of the calmodulin-peptide complex by small angle X-ray scattering. Upon peptide binding, calmodulin lost its dumbbell shape, adopting a more globular conformation. We also studied the energetics of the interaction using calorimetry and computational methods. All our data indicate that calmodulin binds to the peptide from cx43 in the classical 'collapsed' conformation.

Background Multiple profilin isoforms exist in mammals; at least four are expressed in the mammalian testis. The testis-specific isoforms profilin-3 (PFN3) and profilin-4 (PFN4) may have specialized roles in spermatogenic cells which are distinct from known functions fulfilled by the "somatic" profilins, profilin-1 (PFN1) and profilin-2 (PFN2). Results Ligand interactions and spatial distributions of PFN3 and PFN4 were compared by biochemical, molecular and immunological methods; PFN1 and PFN2 were employed as controls. β-actin, phosphoinositides, poly-L-proline and mDia3, but not VASP, were confirmed as in vitro interaction partners of PFN3. In parallel experiments, PFN4 bound to selected phosphoinositides but not to poly-L-proline, proline-rich proteins, or actin. Immunofluorescence microscopy of PFN3 and PFN4 revealed distinct subcellular locations in differentiating spermatids. Both were associated first with the acroplaxome and later with the transient manchette. Predicted 3D structures indicated that PFN3 has the actin-binding site conserved, but retains only approximately half of the common poly-L-proline binding site. PFN4, in comparison, has lost both, polyproline and actin binding sites completely, which is well in line with the experimental data. Conclusion The testis-specific isoform PFN3 showed major hallmarks of the well characterized "somatic" profilin isoforms, albeit with distinct binding affinities. PFN4, on the other hand, did not interact with actin or polyproline in vitro. Rather, it seemed to be specialized for phospholipid binding, possibly providing cellular functions which are distinct from actin dynamics regulation.

A strategy for the accurate determination of protein solution structures starting from X-ray data and a minimal set of NMR data is proposed and successfully applied to two complexes of calmodulin (CaM) with target peptides not previously described. Its implementation in the present case is based on the use of lanthanide ions as substitutes for calcium in one of the four calcium binding sites of CaM and the collection of pseudocontact shift (pcs) and residual dipolar coupling (rdc) restraints induced by the paramagnetic metals. Starting from the crystal structures, new structural models are calculated that are in excellent agreement with the paramagnetic restraints and differ significantly from the starting crystal structures. In particular, in both complexes, a change in orientation of the first helix of the N-terminal CaM domain and of the whole C-terminal domain is observed. The simultaneous use of paramagnetic pcs and rdc restraints has the following crucial advantages: (i) it allows one to assess the possible presence of interdomain conformational freedom, which cannot be detected if the rdc values are derived from external orienting media; (ii) in the absence of significant conformational freedom, the global orientation tensor can be independently and precisely determined from pcs values, which are less sensitive than rdc values to the presence of local structural inaccuracies, and therefore (iii) the relative rearrangement of a domain or a secondary structure element with respect to the metal-bearing domain can be detected.

Collapsin response mediator protein-2 (CRMP-2) plays a crucial role in axonal guidance and neurite outgrowth during neural development and regeneration. We have studied the interaction between calmodulin (CaM) and CRMP-2 and how Ca(2+)/CaM binding modulates the biological functions of CRMP-2. We have shown that CRMP-2 binds to CaM directly in a Ca(2+)-dependent manner. The CaM binding site of CRMP-2 is proposed to reside in the last helix of the folded domain, and in line with this, a synthesized peptide representing this helix bound to CaM. In addition, CaM binding inhibits a homotetrameric assembly of CRMP-2 and attenuates calpainmediated CRMP-2 proteolysis. Furthermore, a CaM antagonist reduces the number and length of process induced by CRMP-2 overexpression in HEK293 cells. Take together, our data suggest that CRMP-2 is a novel CaM-binding protein and that CaM binding may play an important role in regulating CRMP-2 functions.

Profilins are key regulators of the actin microfilament system and in neuronal tissues the profilin 2a isoform is the most abundant and important profilin. The high-resolution crystal structure of rat profilin 2a has been determined in the absence of ligands. By comparing the structure with those of peptide-liganded profilin 2a and unliganded profilin 2b, it can be concluded that the binding site for proline-rich peptides is pre-organized. The C-terminus of profilin 2a is also well ordered in the absence of ligand peptide, in contrast to the 2b isoform which is generated by alternative splicing. Covalent modifications of four cysteine residues were also detected in profilin 2a, as well as a number of other modifications in profilin 2 from rat brain; such modifications could significantly affect the function of profilin. It was also shown that profilin 2a binds to the neuronal protein palladin, including a synthetic palladin peptide; peptides from another profilin ligand, dynamin 1, failed to interact with both profilin 1 and profilin 2a. These results allow a better understanding of the structure-function relationships and ligand binding of mammalian profilin 2a.

Thioesters are more reactive than oxoesters, and thioester chemistry is important for the reaction mechanisms of many enzymes, including the members of the thiolase superfamily, which play roles in both degradative and biosynthetic pathways. In the reaction mechanism of the biosynthetic thiolase, the thioester moieties of acetyl-CoA and the acetylated catalytic cysteine react with each other, forming the product acetoacetyl-CoA. Although a number of studies have been carried out to elucidate the thiolase reaction mechanism at the atomic level, relatively little is known about the factors determining the affinity of thiolases towards their substrates. We have carried out crystallographic studies on the biosynthetic thiolase from Zoogloea ramigera complexed with CoA and three of its synthetic analogues to compare the binding modes of these related compounds. The results show that both the CoA terminal SH group and the side chain SH group of the catalytic Cys89 are crucial for the correct positioning of substrate in the thiolase catalytic pocket. Furthermore, calorimetric assays indicate that the mutation of Cys89 into an alanine significantly decreases the affinity of thiolase towards CoA. Thus, although the sulfur atom of the thioester moiety is important for the reaction mechanism of thioester-dependent enzymes, its specific properties can also affect the affinity and competent mode of binding of the thioester substrates to these enzymes.

The collapsin response mediator protein 2 (CRMP-2) is a central molecule regulating axonal growth cone guidance. It interacts with the cytoskeleton and mediates signals related to myelin-induced axonal growth inhibition. CRMP-2 has also been characterized as a constituent of neurofibrillary tangles in Alzheimer's disease. CD spectroscopy and thermal stability assays using the Thermofluor method indicated that Ca2+ and Mg2+ affect the stability of CRMP-2 and prevent the formation of beta-aggregates upon heating. Gel filtration showed that the presence of Ca2+ or Mg2+ promoted the formation of CRMP-2 homotetramers, and this was further proven by small-angle X-ray scattering experiments, where a 3D solution structure for CRMP-2 was obtained. Previously, we described a crystal structure of human CRMP-2 complexed with calcium. In the present study, we determined the structure of CRMP-2 in the absence of calcium at 1.9 A resolution. When Ca2+ was omitted, crystals could only be grown in the presence of Mg2+ ions. By a proteomic approach, we further identified a number of post-translational modifications in CRMP-2 from rat brain hippocampus and mapped them onto the crystal structure.

Background The myelin sheath is a multilamellar membrane structure wrapped around the axon, enabling the saltatory conduction of nerve impulses in vertebrates. Myelin basic protein, one of the most abundant myelin-specific proteins, is an intrinsically disordered protein that has been shown to bind calmodulin. In this study, we focus on a 19-mer synthetic peptide from the predicted calmodulin-binding segment near the C-terminus of human myelin basic protein. Results The interaction of native human myelin basic protein with calmodulin was confirmed by affinity chromatography. The binding of the myelin basic protein peptide to calmodulin was tested with isothermal titration calorimetry (ITC) in different temperatures, and Kd was observed to be in the low μM range, as previously observed for full-length myelin basic protein. Surface plasmon resonance showed that the peptide bound to calmodulin, and binding was accompanied by a conformational change; furthermore, gel filtration chromatography indicated a decrease in the hydrodynamic radius of calmodulin in the presence of the peptide. NMR spectroscopy was used to map the binding area to reside mainly within the hydrophobic pocket of the C-terminal lobe of calmodulin. The solution structure obtained by small-angle X-ray scattering indicates binding of the myelin basic protein peptide into the interlobal groove of calmodulin, while calmodulin remains in an extended conformation. Conclusion Taken together, our results give a detailed structural insight into the interaction of calmodulin with a C-terminal segment of a major myelin protein, the myelin basic protein. The used 19-mer peptide interacts mainly with the C-terminal lobe of calmodulin, and a conformational change accompanies binding, suggesting a novel mode of calmodulin-target protein interaction. Calmodulin does not collapse and wrap around the peptide tightly; instead, it remains in an extended conformation in the solution structure. The observed affinity can be physiologically relevant, given the high abundance of both binding partners in the nervous system.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency causes episodic ketoacidosis. We encountered a case of siblings in South Africa in whom a novel homozygous mutation (R268H) was found in genomic DNA. Mutant SCOT protein was very faintly detected in their fibroblasts using immunoblot analysis. Transient expression analysis of R268H mutant cDNA at 37 degrees C revealed that the R268H mutant protein was clearly detected, as much as 50% wild-type, together with 40% residual SCOT activities, hence R268H was first regarded as not being a disease-causing mutation. Since no other mutation was identified, R268H mutation was re-evaluated by further transient expression analysis. Accumulation of the R268H mutant protein was revealed to be strongly temperature dependent; residual SCOT activities were calculated to be 59.7%, 34%, and 4%, respectively, in expression at 30 degrees C, 37 degrees C, and 40 degrees C in SV40-transformed fibroblasts of GS01(a homozygote of S283X). SCOT activity of the R268H protein was more vulnerable than the wild-type to heat treatment at 50 degrees C. These results indicated that the R268H mutant protein was clearly more unstable than the wild-type in a temperature-sensitive manner. Furthermore, an analysis of the three-dimensional structure of SCOT showed that the R268H mutation was expected to break a conserved salt bridge between R268 and D52, which would be expected to lead to decreased stability of the protein. Hence we finally concluded that the R268H mutation is a disease-causing one. The stability of mutant protein in transient expression analysis does not always reflect the condition in patients' fibroblasts.

Background The C-terminal tetratricopeptide (TPR) repeat domain of Pex5p recognises proteins carrying a peroxisomal targeting signal type 1 (PTS1) tripeptide in their C-terminus. Previously, structural data have been obtained from the TPR domain of Pex5p in both the liganded and unliganded states, indicating a conformational change taking place upon cargo protein binding. Such a conformational change would be expected to play a major role both during PTS1 protein recognition as well as in cargo release into the peroxisomal lumen. However, little information is available on the factors that may regulate such structural changes. Results We have used a range of biophysical and computational methods to further analyse the conformational flexibility and ligand binding of Pex5p. A new crystal form for the human Pex5p C-terminal domain (Pex5p(C)) was obtained in the presence of Sr2+ ions, and the structure presents a novel conformation, distinct from all previous liganded and apo crystal structures for Pex5p(C). The difference relates to a near-rigid body movement of two halves of the molecule, and this movement is different from that required to reach a ring-like conformation upon PTS1 ligand binding. The bound Sr2+ ion changes the dynamic properties of Pex5p(C) affecting its conformation, possibly by making the Sr2+-binding loop – located near the hinge region for the observed domain motions – more rigid. Conclusion The current data indicate that Pex5p(C) is able to sample a range of conformational states in the absence of bound PTS1 ligand. The domain movements between various apo conformations are distinct from those involved in ligand binding, although the differences between all observed conformations so far can be characterised by the movement of the two halves of Pex5p(C) as near-rigid bodies with respect to each other.

Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the beta barrel and the alpha-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wildtype P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.

2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) is an enzyme highly abundant in the central nervous system myelin of terrestrial vertebrates. The catalytic domain of CNPase belongs to the 2H phosphoesterase superfamily and catalyzes the hydrolysis of nucleoside 2′,3′-cyclic monophosphates to nucleoside 2′-monophosphates. The detailed reaction mechanism and the essential catalytic amino acids involved have been described earlier, but the roles of many amino acids in the vicinity of the active site have remained unknown. Here, several CNPase catalytic domain mutants were studied using enzyme kinetics assays, thermal stability experiments and X-ray crystallography. Additionally, the crystal structure of a perdeuterated CNPase catalytic domain was refined at atomic resolution to obtain a detailed view of the active site and the catalytic mechanism. The results specify determinants of ligand binding and novel essential residues required for CNPase catalysis. For example, the aromatic side chains of Phe235 and Tyr168 are crucial for substrate binding and Arg307 may affect active site electrostatics and regulate loop dynamics. The β5-α7 loop, unique for CNPase in the 2H phosphoesterase family, appears to have various functions in the CNPase reaction mechanism, from coordinating the nucleophilic water molecule to providing a binding pocket for the product and being involved in product release.

Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

Inosine triphosphatase (ITPA) is a ubiquitous key regulator of cellular non-canonical nucleotide levels. It breaks down inosine and xanthine nucleotides generated by deamination of purine bases. Its enzymatic action prevents accumulation of ITP and reduces the risk of incorporation of potentially mutagenic inosine nucleotides into nucleic acids. Here we describe the crystal structure of human ITPA in complex with its prime substrate ITP, as well as the apoenzyme at 2.8 and 1.1A, respectively. These structures show for the first time the site of substrate and Mg2+ coordination as well as the conformational changes accompanying substrate binding in this class of enzymes. Enzyme substrate interactions induce an extensive closure of the nucleotide binding grove, resulting in tight interactions with the base that explain the high substrate specificity of ITPA for inosine and xanthine over the canonical nucleotides. One of the dimer contact sites is made up by a loop that is involved in coordinating the metal ion in the active site. We predict that the ITPA deficiency mutation P32T leads to a shift of this loop that results in a disturbed affinity for nucleotides and/or a reduced catalytic activity in both monomers of the physiological dimer.

The myelin sheath is an insulating membrane layer surrounding myelinated axons in vertebrates, which is formed when the plasma membrane of an oligodendrocyte or a Schwann cell wraps itself around the axon. A large fraction of the total protein in this membrane layer is comprised of only a small number of individual proteins, which have certain intriguing structural properties. The myelin proteins are implicated in a number of neurological diseases, including, for example, autoimmune diseases and peripheral neuropathies. In this review, the structural properties of a number of myelin-specific proteins are described.

Cytidine triphosphate synthetase (CTPS) is a key enzyme in nucleic acid and phospholipid biosynthesis and its activity is increased in certain human cancers, making it a promising drug target. The crystal structure of the synthetase domain of human CTPS, which represents the first structure of a CTPS from an eukaryote, has been determined. The structure is homotetrameric and each active site is formed by three different subunits. Sulfate ions bound to the active sites indicate the positions of phosphate-binding sites for the substrates ATP and UTP and the feedback inhibitor CTP. Together with earlier structures of bacterial CTPS, the human CTPS structure provides an extended understanding of the structure–function relationship of CTPS-family members. The structure also serves as a basis for structure-based design of anti-proliferative inhibitors.

10-Formyltetrahydrofolate dehydrogenase is a ubiquitously expressed enzyme in the human body. It catalyses the formation of tetrahydrofolate and carbon dioxide from 10-­formyltetrahydrofolate, thereby playing an important role in the human metabolism of one-carbon units. It is a two-domain protein in which the N-terminal domain hydrolyses 10-formyltetrahydrofolate into formate and tetrahydrofolate. The high-resolution crystal structure of the hydrolase domain from human 10-formyltetrahydrofolate dehydrogenase has been determined in the presence and absence of a substrate analogue. The structures reveal conformational changes of two loops upon ligand binding, while key active-site residues appear to be pre-organized for catalysis prior to substrate binding. Two water molecules in the structures mark the positions of key oxygen moieties in the catalytic reaction and reaction geometries are proposed based on the structural data.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination and loss of neurological function, local macrophage infiltrate and neuroantigen-specific CD4(+)T cells. MS arises from complex interactions between genetic, immunological, infective and biochemical mechanisms. Although the circumstances of MS etiology remain hypothetical, one persistent theme involves immune system recognition of myelin-specific antigens derived from myelin basic protein, the most abundant extrinsic myelin membrane protein, and/or another equally suitable myelin protein or lipid. Knowledge of the biochemical and physico-chemical properties of myelin proteins and lipids, particularly their composition, organization, structure and accessibility with respect to the compacted myelin multilayers, becomes central to understanding how and why myelin-specific antigens become selected during the development of MS. This review focuses on the current understanding of the molecular basis of MS with emphasis: (i) on the physical-chemical properties, organization, morphology, and accessibility of the proteins and lipids within the myelin multilayers; (ii) on the structure-function relationships and characterization of the myelin proteins relevant to the manifestation and evolution of MS; (iii) on conformational relationships between myelin epitopes which might become selected during the development of MS; (iv) on the structure of MHC/HLA in complex with MBP peptides as well as with TCR, which is crucial to the understanding of the pathogenesis of MS with the ultimate goal of designed antigen-specific treatments. Curr Med Chem

alpha-Methylacyl-CoA racemase is a key enzyme in the metabolism of 2-methyl-branched fatty acids and, in mammals, in the conversion of cholesterol to bile acids. The enzyme from Mycobacterium tuberculosis has been purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method. The crystals of the unliganded racemase belong to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 122.0, c = 256.4 A. Data sets were collected at 100 K. The crystals diffract to 2.8 A using synchrotron radiation.

Biosynthetic thiolase catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA. This is a key step in the synthesis of many biological compounds, including steroid hormones and ketone bodies. The thiolase reaction involves two chemically distinct steps; during acyl transfer, an acetyl group is transferred from acetyl-CoA to Cys89, and in the Claisen condensation step, this acetyl group is further transferred to a second molecule of acetyl-CoA, generating acetoacetyl-CoA. Here, new crystallographic data for Zoogloea ramigera biosynthetic thiolase are presented, covering all intermediates of the thiolase catalytic cycle. The high-resolution structures indicate that the acetyl group goes through four conformations while being transferred from acetyl-CoA via the acetylated enzyme to acetoacetyl-CoA. This transfer is catalyzed in a rigid cavity lined by mostly hydrophobic side chains, in addition to the catalytic residues Cys89, His348, and Cys378. The structures highlight the importance of an oxyanion hole formed by a water molecule and His348 in stabilizing the negative charge on the thioester oxygen atom of acetyl-CoA at two different steps of the reaction cycle. Another oxyanion hole, composed of the main chain nitrogen atoms of Cys89 and Gly380, complements a negative charge of the thioester oxygen anion of the acetylated intermediate, stabilizing the tetrahedral transition state of the Claisen condensation step. The reactivity of the active site may be modulated by hydrogen bonding networks extending from the active site toward the back of the molecule.

Myelin, the multilayered membrane structure surrounding axons, provides a unique environment to its proteins, which are either transmembrane proteins or interacting intimately with the membrane surface. Although myelin-specific proteins have been studied for decades, remarkably little is known of their three-dimensional structures. In addition, the exact functions of myelin proteins are to a large extent unknown. In this report, our current knowledge of peripheral nervous system myelin protein structures is reviewed, and the current status of attempts to solve the structures of full-length myelin proteins is evaluated. Furthermore, molecular models for the extracellular domain of the myelin-associated glycoprotein and the putative kinase-like domain of 2',3'-cyclic nucleotide 3'-phosphodiesterase are presented and discussed.

The myelin-associated glycoprotein is a transmembrane cell adhesion molecule expressed specifically by myelinating glial cells of the nervous system. Its two isoforms, whose amino acid sequences differ only by their respective cytoplasmic carboxy-terminal domains, are important for the formation and maintenance of a normal functional myelin sheath. In this study, by using recombinant proteins, we identify the cytoplasmic domain of the small isoform of the myelin-associated glycoprotein as a zinc-binding protein. The observed dissociation constant lies in the low micromolar range (K(D) = 6-7 microM). The binding of zinc by the small myelin-associated glycoprotein induces a conformational change that enables the protein to reversibly bind to a hydrophobic phenyl-Sepharose matrix. Our results also suggest that zinc may induce dimerization of the small myelin-associated glycoprotein. We suggest roles for zinc in the stabilization of the structure of the cytoplasmic domain of the small myelin-associated glycoprotein and in protein-protein interactions that involve this short domain.

The myelin-associated glycoprotein is a transmembrane cell adhesion molecule expressed by myelinating glial cells of the nervous system. So far, only protein kinases have been reported to interact with the cytoplasmic domains of the two isoforms of the myelin-associated glycoprotein. We report here the identification of the first nonkinase intracellular ligand for the large isoform of the myelin-associated glycoprotein as the S100beta protein. The interaction is dependent on the presence of calcium. We have also localized the S100beta-binding site in the cytoplasmic domain specific to the large myelin-associated glycoprotein isoform to a putative basic amphipathic alpha-helix. A synthetic peptide corresponding to this region bound to S100beta in a calcium-dependent manner with a stoichiometric ratio of 1:1 (K(D) approximately 7 microM). We suggest that the observed interaction may play a role in the regulation of the myelinating glial cell cytoskeleton and the divalent cation-dependent signal transduction events during myelin formation and maintenance.

The dynamics of reconstituted myelin membranes were studied by incoherent neutron scattering when the myelin basic protein was added. The mutant C8 has properties similar to the highly modified form seen in Multiple Sclerosis patients and tends to bring the membrane in a more flexible state. This might be an important element to shed light on the associated pathology.

As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids - a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.

The myelin sheath is a tightly packed multilayered membrane structure insulating selected axons in the central and the peripheral nervous systems. Myelin is a biochemically unique membrane, containing a specific set of proteins. In this study, we expressed and purified recombinant human myelin P2 protein and determined its crystal structure to a resolution of 1.85 A. A fatty acid molecule, modeled as palmitate based on the electron density, was bound inside the barrel-shaped protein. Solution studies using synchrotron radiation indicate that the crystal structure is similar to the structure of the protein in solution. Docking experiments using the high-resolution crystal structure identified cholesterol, one of the most abundant lipids in myelin, as a possible ligand for P2, a hypothesis that was proven by fluorescence spectroscopy. In addition, electrostatic potential surface calculations supported a structural role for P2 inside the myelin membrane. The potential membrane-binding properties of P2 and a peptide derived from its N terminus were studied. Our results provide an enhanced view into the structure and function of the P2 protein from human myelin, which is able to bind both monomeric lipids inside its cavity and membrane surfaces.

The myelin sheath is a multilayered, tightly packed membrane wrapped around axons, enabling the rapid saltatory transmission of nerve impulses. Myelin carries a set of specific proteins, most of which are either integral or peripheral membrane proteins. In the peripheral nervous system, one of the myelin-specific proteins is P2, a member of the fatty acid binding protein (FABP) family that binds monomeric lipids inside its barrel structure. P2 is a peripheral membrane protein, and in addition to binding membrane surfaces, it is able to stack lipid bilayers. We performed a detailed structure-function analysis of recombinant human peripheral nerve P2. The liganded crystal structure of the wild-type human P2 protein was determined at 0.93-A resolution, allowing a very detailed analysis of fatty acid binding. In addition, 14 structure-based mutants were produced and characterized with respect to crystal and solution structure as well as membrane interactions and lipid binding. While the surface charge distribution of P2 presents two positively charged faces as putative membrane binding sites, the mutations with largest effects on P2 function did not affect the basic surface residues. Instead, it is likely they affect the dynamics and structure of the helical lid domain, which is expected to open and close upon lipid binding and to partially insert into the membrane. Furthermore, a hinge domain mutant could be crystallized in the absence of bound lipid. We also show that P2 gets oriented when binding to membrane surfaces. Overall, our current data allow a very detailed understanding of the structure-function relationships in myelin P2, a unique FABP, which is able to promote membrane multilayer formation.