Supplementary MaterialsDatapack S1: Standalone iSee datapack – provides the enhanced version of this article for use offline. monomer and higher-order oligomer in equilibrium with the dimer. Crystal packing analysis reveals the existence of a crystallographic hexamer, and that the kinase domain dimerizes through the C-lobe subdomain. Mapping of disease-related missense mutations onto the kinase domain structure revealed that the mutation sites could be classified into four different groups based on the location C dimer interface, interlobar helices, protein surface, or within other secondary structural elements. Conclusions The crystal structure of the kinase domain of GNE provides a structural basis for understanding disease-causing mutations and a model of hexameric wild type full length enzyme. Enhanced Version This article can also be viewed as an enhanced version in which the text of the article is usually integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. Introduction Sialic acids are N- or substituted terminal monosaccharides with a nine-carbon backbone highly expressed on eukaryotic cell surfaces [1]. Sialylation of glycoproteins and glycolipids modulates a wide range of biological and pathological events including early development [2], tumorigenesis [3], viral and bacterial infection, and immunity [4], [5]. In vertebrate systems, N-acetylneuraminic acid (Neu5Ac) is the metabolic precursor of all known naturally occurring sialic acids [6]. Neu5Ac is usually synthesized in the cytosol from UDP-N-acetylglucosamine (UDP-GlcNAc) by four consecutive reactions; and UDP-GlcNAc is usually a derivative of fructose-6-phosphate and the end-product of the hexosamine biosynthesis pathway (Physique 1). Open in a separate window Figure 1 Key sugar molecules in the sialic acid biosynthesis pathway. The first two actions of the biosynthesis of Neu5Ac from UDP-GlcNAc are catalyzed by the bi-functional enzyme UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE). GNE contains an N-terminal epimerase domain and a C-terminal kinase domain [7]. The TGX-221 manufacturer epimerase domain converts UDP-GlcNAc to N-acetylmannosamine (ManNAc), which is TGX-221 manufacturer then phosphorylated at the 6 position by the kinase domain. GNE is usually feedback-inhibited by Rabbit Polyclonal to COX41 the activated form of Neu5Ac, i.e., cytidine-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). The kinase domain belongs to the ROK (Repressor, ORF, Kinase) family. The ROK family consists of a set of bacterial proteins that include repressors for sugar catabolic operons, and sugar kinases [8]. is the only known gene in the entire human genome that encodes a ROK domain-containing protein. Three protein isoforms have been explained for human GNE, where isoform 1 is usually ubiquitously expressed and is usually believed to be responsible for the basic supply of sialic acids. Isoforms 2 and 3 are generated by option splicing and show tissue specific expression patterns. Isoforms 2 and 3 have reduced epimerase activities but almost intact kinase activities and may fine-tune the production of sialic acids [9]. Wild type GNE forms homo-hexamer in answer [10], and allosteric regulation of the epimerase and kinase activities of GNE is usually important for the normal function of TGX-221 manufacturer the protein [10], [11]. Mutations in the epimerase domain lead to the rare congenital metabolism disorder sialurea, which results in the production of high levels of Neu5Ac due to loss of the allosteric feedback control of the UDP-GlcNAc 2-epimerase activity by CMP-Neu5Ac [12]. Late onset autosomal recessive inclusion body myopathy, which is also known as hereditary inclusion body myopathy (hereinafter referred to as HIBM), and allelic Nonaka myopathy are neuromuscular disorders that are caused by a number of different mutations within the gene. The mutations are located at either the epimerase domain or the kinase domain [13] and lead to hypoactivity of the enzyme [11]. Mutagenesis and enzymatic activity analysis revealed that the activities of the epimerase domain and the kinase domain are interrelated such that a single mutation in one domain could impact the activities of both domains [11]. Here, we solved the structure of the dimeric GNE kinase domain in the ligand-free state. The structure reveals the dimerization interface of the kinase domain and also suggests a possible hexameric assembly of the protein. Furthermore, the structure provides insights into the relationship between GNE mutations and GNE-related metabolism disorders. Results and Discussion Overview of the GNE kinase domain monomer The overall structure adopts.