¹H, ¹⁵N and ¹³C NMR Assignments, Secondary Structure and Overall Topology of the Escherichia Coll Glgs Protein

Abstract

GlgS is a 7892-Da protein which is involved in glycogen biosynthesis in bacteria. We report the 'H, "N and 13C NMR assignments of the backbone and side-chain resonances at 25 "C and pH 6.7 from twodimensional homonuclear and three-dimensional heteronuclear NMR experiments. The secondary structure of the protein was determined using sequential and medium-range NOE correlations, vicinal 3.fNH-Ha coupling values and amide proton exchange rates. The secondary structure obtained is consistent with the secondary chemical shifts of 'Ha, I3Ca and "C = 0. It was found that the secondary structure of GlgS comprises two amphipathic helices (AsnlO-Met21 and Glu39 -Arg60), one short highly hydrophobic helix (Ile30-Va133), a short extended P-strand-like fragment (Arg26-Asp29) and two type I jj-turns (His22-Gly25 and Thr34-Met37). An overall topology of GlgS is suggested based on long-range NOES. The elements of secondary structure form a sandwich in which the P-strand and the short hydrophobic helix are positioned between the two amphipathic helices.Keywords: protein structure ; NMR; glycogen; heteronuclear NMR; GlgS protein.In response to different environmental stresses such as starvation, high osmolarity, high or low temperature, Escherichia coli cells undergo considerable physiological changes. Via the stress-associated sigma factor g s (encoded by the rpoS gene) (Loewen and Hengge-Aronis, 1994), the expression of a number of stress-specific 0'-dependent genes is induced. The cS protein acts by modifying the promoter recognition of the RNA polymerase complex. Starvation activates the genes for glycogen biosynthesis. When the growth medium is exhausted for an essential nutrient(s), E. coli cells start the transition into stationary phase and accumulate glycogen if glucose or other carbon sources are still present (Okita et al., 1981).Glycogen synthesis in bacteria occurs via the ADP-glucose pathway. Three reactions are involved. First, ADP-glucose is synthesised in a reaction catalysed by ADP-glucose pyrophosphorylase (glgC gene). Then, catalysed by ADP-glucose-specific glycogen synthase (glgA gene), a new a-1,4-glycosidic bond is formed. Subsequently, the branching enzyme (glgB gene) catalyses formation of the branched a-I ,6-glycosidic linkages in glycogen. These three structural genes are organised in the glg gene cluster at 75 min on the E. coli chromosome. The enzymatic reactions have been extensively studied from the point of regulation of enzyme activity and expression of the glycogen biosynthetic enzymes (reviewed by Preiss and Romeo, 1993). It has been shown that the 0 ' sigma factor is required for glycogen accumulation (Lange and Hengge-Aronis, 1991) but it does not regulate transcription of the glgC and glgA genes (HenggeAronis and Fischer, 1992). Using a o* promoter screen, a novel gene induced by 0 ' and encoding a protein involved in the glycogen synthesis was discovered and cloned (Hengge-Aronis and Fischer, 1992). This gene is situated at 66.6 min (3247 kb) on the E. coli chromosome outside of the glg ge...

Regulation of glycogen metabolism in yeast and bacteria

“…lacZ fusion analyses on WT and rpoS mutant cells have shown that RpoS does not regulate glgCAP transcription in E. coli (Hengge-Aronis & Fischer, 1992;Montero et al, 2009), but positively controls the expression of glgS, a gene whose product exerts a positive, but still undefined effect on glycogen accumulation (Hengge-Aronis & Fischer, 1992;Eydallin et al, 2010;Montero et al, 2009). This gene codes for a 7.9-kDa protein, which is hydrophilic, highly charged and has no significant sequence similarity to any other protein present in databases outside enterobacteria (Beglova et al, 1997;Kozlov et al, 2004). Sequence analyses of the 1000-bp-long promoter region upstream from the ATG initiation codon of glgS did not reveal the presence of a putative RpoS box, defined in E. coli as TGN 0 À 2 CYATAMT (Lacour & Landini, 2004) or TCTATACTTAA (Weber et al, 2005).…”

Structure of GlgS from Escherichia coli suggests a role in protein–protein interactions

“…The most structured region of GlgS is the fragment between Asn6 and Met57. The five N-terminal and eight C-terminal amides give low heteronuclear NOE values indicating high mobility and an unstructured C-terminus [12]. …”

“…Two single-residue mutations S17G and F45S abolish the ability of GlgS to stimulate glycogen accumulation [12]. Phe45 is a part of the hydrophobic core and its substitution with a hydrophilic serine residue is likely to disrupt the protein tertiary structure.…”

“…Earlier work suggested that GlgS might be a site for primary sugar attachment in the glycogen synthesis pathway [12]. This hypothesis has been weakened by the recent finding that glycogen synthase in Agrobacterium tumefaciens does not require additional proteins for glycogen priming [6].…”

“…NMR experiments were performed at 298 K. Backbone and side-chain NMR signal assignments of GlgS were determined previously [12]. NOE constraints for the structure determination were obtained from 15 N-edited and homonuclear NOESY obtained on a Bruker Avance600 MHz spectrometer.…”