Search

Background The AHNAK2 gene encodes a large nucleoprotein expressed in several tissues, including brain, squamous epithelia, smooth muscle, and neuropil. Its role in calcium signaling has been suggested and to date, clear evidence about its involvement in the pathogenesis of clinical disorders is still lacking. Methods Here, we report a female 24-year-old patient diagnosed with a cardio-facio-cutaneous-like phenotype (CFC-like), characterized by epilepsy, psychomotor development delay, atopic dermatitis, congenital heart disease, hypotonia, and facial dysmorphism, who is compound heterozygote for two missense mutations in the AHNAK2 gene detected by exome sequencing. Results This patient had no detectable variant in any of the genes known to be associated with the cardio-facio-cutaneous syndrome. Moreover, the mode of inheritance does not appear to be autosomal dominant, as it is in typical CFC syndrome. We have performed in silico assessment of mutation severity separately for each missense mutation, but this analysis excludes a severe effect on protein function. Protein structure predictions indicate the mutations are located in flexible regions possibly involved in molecular interactions. Conclusion We discuss an alternative interpretation on the potential involvement of the two missense mutations in the AHNAK2 gene on the expression of CFC-like phenotype in this patient based on inter-allelic complementation. publishedVersion

N-myc downstream-regulated gene 1 (NDRG1) is a tumour suppressor involved in vesicular trafficking and stress response. NDRG1 participates in peripheral nerve myelination, and mutations in the NDRG1 gene lead to Charcot-Marie-Tooth neuropathy. The 43-kDa NDRG1 is considered as an inactive member of the α/β hydrolase superfamily. In addition to a central α/β hydrolase fold domain, NDRG1 consists of a short N terminus and a C-terminal region with three 10-residue repeats. We determined the crystal structure of the α/β hydrolase domain of human NDRG1 and characterised the structure and dynamics of full-length NDRG1. The structure of the α/β hydrolase domain resembles the canonical α/β hydrolase fold with a central β sheet surrounded by α helices. Small-angle X-ray scattering and CD spectroscopy indicated a variable conformation for the N- and C-terminal regions. NDRG1 binds to various types of lipid vesicles, and the conformation of the C-terminal region is modulated upon lipid interaction. Intriguingly, NDRG1 interacts with metal ions, such as nickel, but is prone to aggregation in their presence. Our results uncover the structural and dynamic features of NDRG1, as well as elucidate its interactions with metals and lipids, and encourage studies to identify a putative hydrolase activity of NDRG1. DATABASES: The coordinates and structure factors for the crystal structure of human NDRG1 were deposited to PDB (PDB ID: 6ZMM).

The activity-regulated cytoskeleton-associated protein (ARC) is critical for long-term synaptic plasticity and memory formation. Acting as a protein interaction hub, ARC regulates diverse signalling events in postsynaptic neurons. A protein interaction site is present in the ARC C-terminal domain (CTD), a bilobar structure homologous to the retroviral Gag capsid domain. We hypothesized that detailed knowledge of the three-dimensional molecular structure of monomeric full-length ARC is crucial to understand its function; therefore, we set out to determine the structure of ARC to understand its various functional modalities. We purified recombinant ARC and analyzed its structure using small-angle X-ray scattering and synchrotron radiation circular dichroism spectroscopy. Monomeric full-length ARC has a compact, closed structure, in which the oppositely charged N-terminal domain (NTD) and CTD are juxtaposed, and the flexible linker between them is not extended. The modeled structure of ARC is supported by intramolecular live-cell Förster resonance energy transfer imaging in rat hippocampal slices. Peptides from several postsynaptic proteins, including stargazin, bind to the N-lobe, but not to the C-lobe, of the bilobar CTD. This interaction does not induce large-scale conformational changes in the CTD or flanking unfolded regions. The ARC NTD contains long helices, predicted to form an anti-parallel coiled coil; binding of ARC to phospholipid membranes requires the NTD. Our data support a role for the ARC NTD in oligomerization as well as lipid membrane binding. The findings have important implications for the structural organization of ARC with respect to distinct functions, such as postsynaptic signal transduction and virus-like capsid formation. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

The Shank proteins are crucial scaffolding elements of the post-synaptic density (PSD). One of the best-characterized domains in Shank is the PDZ domain, which binds to C-terminal segments of several other PSD proteins. We carried out a detailed structural analysis of Shank3 PDZ domain-peptide complexes, to understand determinants of binding affinity towards different ligand proteins. Ligand peptides from four different proteins were cocrystallized with the Shank3 PDZ domain, and binding affinities were determined calorimetrically. In addition to conserved class I interactions between the first and third C-terminal peptide residue and Shank3, side chain interactions of other residues in the peptide with the PDZ domain are important factors in defining affinity. Structural conservation suggests that the binding specificities of the PDZ domains from different Shanks are similar. Two conserved buried water molecules in PDZ domains may affect correct local folding of ligand recognition determinants. The solution structure of a tandem Shank3 construct containing the SH3 and PDZ domains showed that the two domains are close to each other, which could be of relevance, when recognizing and binding full target proteins. The SH3 domain did not affect the affinity of the PDZ domain towards short target peptides, and the schizophrenia-linked Shank3 mutation R536W in the linker between the domains had no effect on the structure or peptide interactions of the Shank3 SH3-PDZ unit. Our data show the spatial arrangement of two adjacent Shank domains and pinpoint affinity determinants for short PDZ domain ligands with limited sequence homology.

The catalytic domain of the trimeric human Δ(3),Δ(2)-enoyl-CoA isomerase, type 2 (HsECI2), has the typical crotonase fold. In the active site of this fold two main chain NH groups form an oxyanion hole for binding the thioester oxygen of the 3E- or 3Z-enoyl-CoA substrate molecules. A catalytic glutamate is essential for the proton transfer between the substrate C2 and C4 atoms for forming the product 2E-enoyl-CoA, which is a key intermediate in the β-oxidation pathway. The active site is covered by the C-terminal helix-10. In HsECI2, the isomerase domain is extended at its N terminus by an acyl-CoA binding protein (ACBP) domain. Small angle X-ray scattering analysis of HsECI2 shows that the ACBP domain protrudes out of the central isomerase trimer. X-ray crystallography of the isomerase domain trimer identifies the active site geometry. A tunnel, shaped by loop-2 and extending from the catalytic site to bulk solvent, suggests a likely mode of binding of the fatty acyl chains. Calorimetry data show that the separately expressed ACBP and isomerase domains bind tightly to fatty acyl-CoA molecules. The truncated isomerase variant (without ACBP domain) has significant enoyl-CoA isomerase activity; however, the full-length isomerase is more efficient. Structural enzymological studies of helix-10 variants show the importance of this helix for efficient catalysis. Its hydrophobic side chains, together with residues from loop-2 and loop-4, complete a hydrophobic cluster that covers the active site, thereby fixing the thioester moiety in a mode of binding competent for efficient catalysis.

Peripheral myelin protein 2 (Pmp2, P2 or Fabp8), a member of the fatty acid binding protein family, was originally described together with myelin basic protein (Mbp or P1) and myelin protein zero (Mpz or P0) as one of the most abundant myelin proteins in the peripheral nervous system (PNS). Although Pmp2 is predominantly expressed in myelinated Schwann cells, its role in glia is currently unknown. To study its function in PNS biology, we have generated a complete Pmp2 knockout mouse (Pmp2(-/-) ). Comprehensive characterization of Pmp2(-/-) mice revealed a temporary reduction in their motor nerve conduction velocity (MNCV). While this change was not accompanied by any defects in general myelin structure, we detected transitory alterations in the myelin lipid profile of Pmp2(-/-) mice. It was previously proposed that Pmp2 and Mbp have comparable functions in the PNS suggesting that the presence of Mbp can partially mask the Pmp2(-/-) phenotype. Indeed, we found that Mbp lacking Shi(-/-) mice, similar to Pmp2(-/-) animals, have preserved myelin structure and reduced MNCV, but this phenotype was not aggravated in Pmp2(-/-) /Shi(-/-) mutants indicating that Pmp2 and Mbp do not substitute each other's functions in the PNS. These data, together with our observation that Pmp2 binds and transports fatty acids to membranes, uncover a role for Pmp2 in lipid homeostasis of myelinating Schwann cells.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency causes episodic ketoacidotic crises and no apparent symptoms between them. Here, we report a Japanese case of neonatal-onset SCOT deficiency. The male patient presented a severe ketoacidotic crisis, with blood pH of 7.072 and bicarbonate of 5.8 mmol/L at the age of 2 days and was successfully treated with intravenous infusion of glucose and sodium bicarbonate. He was diagnosed as SCOT deficient by enzymatic assay and mutation analysis. At the age of 7 months, he developed a second ketoacidotic crisis, with blood pH of 7.059, bicarbonate of 5.4 mmol/L, and total ketone bodies of 29.1 mmol/L. He experienced two milder ketoacidotic crises at the ages of 1 year and 7 months and 3 years and 7 months. His urinary ketone bodies usually range from negative to 1+ but sometimes show 3+ (ketostix) without any symptoms. Hence, this patient does not show permanent ketonuria, which is characteristic of typical SCOT-deficient patients. He is a compound heterozygote of c.1304C > A (T435N) and c.658-666dupAACGTGATT p.N220_I222dup. mutations in the OXCT1 gene. The T435N mutation was previously reported as one which retained significant residual activity. The latter novel mutation was revealed to retain no residual activity by transient expression analysis. Both T435N and N220_I222 lie close to the SCOT dimerization interface and are not directly connected to the active site in the tertiary structure of a human SCOT dimer. In transient expression analysis, no apparent interallelic complementation or dominant negative effects were observed. Significant residual activity from the T435N mutant allele may prevent the patient from developing permanent ketonuria.

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.

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.

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.