Abstract : The myelin-associated glycoprotein is a transmembrane cell adhesion molecule expressed... more Abstract : 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 myelinassociated glycoprotein. We report here the identification of the first nonkinase intracellular ligand for the large isoform of the myelin-associated glycoprotein as the S100β protein. The interaction is dependent on the presence of calcium. We have also localized the S100β-binding site in the cytoplasmic domain specific to the large myelin-associated glycoprotein isoform to a putative basic amphipathic α-helix. A synthetic peptide corresponding to this region bound to S100β in a calcium-dependent manner with a stoichiometric ratio of 1:1 (KD≈ 7 μM). 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.
Schistosoma japonicum glutathione S-transferase (SjGST) is a common fusion tag in recombinant pro... more Schistosoma japonicum glutathione S-transferase (SjGST) is a common fusion tag in recombinant protein production, and its 3-dimensional structure has been studied in the context of drug design. We have determined the crystal structure of non-fused SjGST complexed with glutathione, and compare it to complexes between glutathione and SjGST fusion proteins.
Biosynthetic thiolase catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA... more 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.
Collapsin response mediator protein-2 (CRMP-2) plays a crucial role in axonal guidance and neurit... more 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 Ca2+/CaM binding modulates the biological functions of CRMP-2. We have shown that CRMP-2 binds to CaM directly in a Ca2+-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.
Inosine triphosphatase (ITPA) is a ubiquitous key regulator of cellular non-canonical nucleotide ... more 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.
Axonal growth cone guidance is a central process in nervous system development and repair. Collap... more Axonal growth cone guidance is a central process in nervous system development and repair. Collapsin response mediator protein 2 (CRMP-2) is a neurite extension-promoting neuronal cytosolic molecule involved in the signalling of growth inhibitory cues from external stimuli, such as semaphorin 3A and the myelin-associated glycoprotein. We have determined the crystal structure of human tetrameric CRMP-2, which is structurally related to the dihydropyriminidases; however, the active site is not conserved. The wealth of earlier functional mapping data for CRMP-2 are discussed in light of the three-dimensional structure of the protein. The differences in oligomerisation interfaces between CRMP-1 and CRMP-2 are used to model CRMP-1/2 heterotetramers.
Abstract : The myelin-associated glycoprotein is a transmembrane cell adhesion molecule expressed... more Abstract : 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 myelinassociated glycoprotein. We report here the identification of the first nonkinase intracellular ligand for the large isoform of the myelin-associated glycoprotein as the S100β protein. The interaction is dependent on the presence of calcium. We have also localized the S100β-binding site in the cytoplasmic domain specific to the large myelin-associated glycoprotein isoform to a putative basic amphipathic α-helix. A synthetic peptide corresponding to this region bound to S100β in a calcium-dependent manner with a stoichiometric ratio of 1:1 (KD≈ 7 μM). 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.
Schistosoma japonicum glutathione S-transferase (SjGST) is a common fusion tag in recombinant pro... more Schistosoma japonicum glutathione S-transferase (SjGST) is a common fusion tag in recombinant protein production, and its 3-dimensional structure has been studied in the context of drug design. We have determined the crystal structure of non-fused SjGST complexed with glutathione, and compare it to complexes between glutathione and SjGST fusion proteins.
Biosynthetic thiolase catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA... more 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.
Collapsin response mediator protein-2 (CRMP-2) plays a crucial role in axonal guidance and neurit... more 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 Ca2+/CaM binding modulates the biological functions of CRMP-2. We have shown that CRMP-2 binds to CaM directly in a Ca2+-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.
Inosine triphosphatase (ITPA) is a ubiquitous key regulator of cellular non-canonical nucleotide ... more 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.
Axonal growth cone guidance is a central process in nervous system development and repair. Collap... more Axonal growth cone guidance is a central process in nervous system development and repair. Collapsin response mediator protein 2 (CRMP-2) is a neurite extension-promoting neuronal cytosolic molecule involved in the signalling of growth inhibitory cues from external stimuli, such as semaphorin 3A and the myelin-associated glycoprotein. We have determined the crystal structure of human tetrameric CRMP-2, which is structurally related to the dihydropyriminidases; however, the active site is not conserved. The wealth of earlier functional mapping data for CRMP-2 are discussed in light of the three-dimensional structure of the protein. The differences in oligomerisation interfaces between CRMP-1 and CRMP-2 are used to model CRMP-1/2 heterotetramers.
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