ABSTRACT␣-Glucan phosphorylases contribute to degradation of glycogen and maltodextrins formed in the course of maltose metabolism in bacteria. Accordingly, bacterial ␣-glucan phosphorylases are classified as either glycogen or maltodextrin phosphorylase, GlgP or MalP, respectively. GlgP and MalP enzymes follow the same catalytic mechanism, and thus their substrate spectra overlap; however, they differ in their regulation: GlgP genes are constitutively expressed and the enzymes are controlled on the activity level, whereas expression of MalP genes are transcriptionally controlled in response to the carbon source used for cultivation. We characterize here the modes of control of the ␣-glucan phosphorylase MalP of the Gram-positive Corynebacterium glutamicum. In accordance to the proposed function of the malP gene product as MalP, we found transcription of malP to be regulated in response to the carbon source. Moreover, malP transcription is shown to depend on the growth phase and to occur independently of the cell glycogen content. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. Since the latter is considered a typical feature of GlgPs, we propose that C. glutamicum MalP acts as both maltodextrin and glycogen phosphorylase and, based on these findings, we question the current system for classification of bacterial ␣-glucan phosphorylases. IMPORTANCEBacterial ␣-glucan phosphorylases have been classified conferring to their purpose as either glycogen or maltodextrin phosphorylases. We found transcription of malP in C. glutamicum to be regulated in response to the carbon source, which is recognized as typical for maltodextrin phosphorylases. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. The latter is considered a typical feature of GlgPs. These findings, taken together, suggest that C. glutamicum MalP is the first ␣-glucan phosphorylase that does not fit into the current system for classification of bacterial ␣-glucan phosphorylases and exemplifies the complex mechanisms underlying the control of glycogen content and maltose metabolism in this model organism.T he ␣-glucan phosphorylases (EC 2.4.1.1) catalyze the reversible cleavage of ␣-1,4-glycosidic linkages in polysaccharides, thereby liberating ␣-glucose-1-phosphate. By this means, ␣-glucan phosphorylases participate in metabolic processes such as degradation of the intracellular storage polysaccharides glycogen and starch (1, 2), as well as the degradation of maltodextrins formed both in the course of the maltose metabolism of various bacteria (3, 4) and in the cytosol of plant leaves (5-7). Several ␣-glucan phosphorylases have been extensively studied, their crystal structures have been solved, and the catalytic mechanisms have been described (8-11). Although the catalytic mechanisms appear to be similar in all hitherto characterized phosphorylases (12-14)...

Pentameric ligand-gated ion channels represent a large family of receptors comprising an extracellular domain, four transmembrane helices and a cytosolic intracellular domain (ICD). ICDs play important roles in receptor localization and trafficking, thus regulating synaptic activity and plasticity. Glycine and GABA type A receptor ICDs bind to the scaffolding protein gephyrin, a master regulator of inhibitory synapses. Here we report the use of yeast lumazine synthase as soluble pentameric protein scaffold for the study of receptor ICDs derived from GlyR alpha1 and beta-subunits. We were able to create ICDs assemblies in a homo- (LS-betaICD) and hetero-pentameric state (LS-alpha/betaICD) and provide first-in-class structural insights on their high structural flexibility using small angle X-ray scattering. We report a high-affinity interaction between the LS-alpha/betaICD and gephyrin leading to the in vitro formation of high-molecular mega-Dalton complexes composed of three gephyrin trimers and three pentamers as basic building block. Depending on the stoichiometric ratios between gephyrin and LS-ICDs the formed complexes grow or shrink in size. In cells, LS-ICDs efficiently recruited gephyrin and were able to accumulate gephyrin at GABAergic synapses in neurons. Our findings collectively propose a new, potentially general, mechanistic concept for a gephyrin-dependent bridging of GlyRs at the inhibitory synapse.

Synaptic inhibition is essential for shaping the dynamics of neuronal networks, and aberrant inhibition is linked to epilepsy. Gephyrin (Geph) is the principal scaffolding protein at inhibitory synapses and is essential for postsynaptic clustering of glycine (GlyRs) and GABA type A receptors (GABAARs). Consequently, gephyrin is crucial for maintaining the relationship between excitation and inhibition in normal brain function and mutations in the gephyrin gene (GPHN) are associated with neurodevelopmental disorders and epilepsy. We identified bi-allelic variants in the GPHN gene, namely the missense mutation c.1264G > A and splice acceptor variant c.1315-2A > G, in a patient with developmental and epileptic encephalopathy (DEE). We demonstrate that the splice acceptor variant leads to nonsense-mediated mRNA decay (NMD). Furthermore, the missense variant (D422N) alters gephyrin structure, as examined by analytical size exclusion chromatography and CD-spectroscopy, thus leading to reduced receptor clustering and sensitivity towards calpain-mediated cleavage. Additionally, both alterations contribute to an observed reduction of inhibitory signal transmission in neurons, which likely contributes to the pathological encephalopathy.