Crystal structures of adenylylated and unadenylylated PII protein GlnK from Corynebacterium glutamicum

The crystal structures of adenylylated and unadenylylated GlnK, a PII protein from Corynebacterium glutamicum, indicate that adenylylation of Tyr51 in the T-loop does not interfere with the PII-typical conformational changes that occur in the T-loop upon effector binding. Rather, T-loop adenylylation further expands the repertoire of mechanisms that enable PII function.


Figure S1
Peptide coverage of adGlnK as determined by mass spectrometry with the adenylylation shown in yellow. The depiction of peptides is not quantitative. The adenylylation rate was estimated to be close to 99%.

Figure S2
Structure-based annotated sequence alignment of GlnK from C. glutanicum and of the ten closest structurally homologous PII proteins as identified with program DALI (see also Supplementary Table S2). Residues explicitly named in the main text are colored as follows: in green, residues interacting with the adenylylated Tyr51 residue; in blue, a residue involved in stabilizing the hexameric assembly via inter-subunit main chain hydrogen bonds and in red, residues involved in phosphate, AMP or ADP binding. Residue numbering for protein data bank entry 2o66 corresponds to that of UNIPROT entry Q9ZST4. It differs form that used in the deposited coordinate file for entry 2o66.

Figure S3
Sequence conservation analyses as performed with program WebLogo 3 (http://weblogo.threeplusone.com/create.cgi; (Crooks et al., 2004)) (a) Weblogo analysis of the alignment of the eleven sequences displayed in Supplementary Fig. S2. Selected residues are highlighted using a very similar color coding as in Supplementary Fig. S2 (b) Weblogo analysis/representation of a multiple sequence alignment of 197 PII proteins identified with ProtBLAST/PSI-BLAST (Camacho et al., 2009). The degree of conservation is reflected in character size. In both panels, residue numbering adheres to the amino acid sequence numbering of GlnK from C. glutamicum.

Figure S4
Interaction plots of the phosphate ions in the ATP-binding pockets towards the C-termini of chains (a) A and C, (b) A and B and (c) B and C generated with program Ligplot+ (Laskowski & Swindells, 2011). Hydrogen bonds are shown as dotted green lines. Residues, which participate in hydrophobic interactions, are highlighted by red circle segments.

Figure S5
Interaction plots of the adenylylated tyrosine residues in (a) chain A, (b) chain B and (c) chain E generated with program Ligplot+ (Laskowski & Swindells, 2011). Covalent bonds are shown as solid purple lines, and hydrogen bonds are shown as dotted green lines. Residues, which participate in hydrophobic interactions with the ligand, are indicated by red circle segments.

Figure S6
Detailed view of (a) the ATP-binding pocket located between chains B (colored green) and C (colored beige) of one adGlnK trimer with the bound AMP molecule shown in blue and (b) the ATP-binding pocket located between chains F (colored orange) and G (colored gray) of the second adGlnK trimer with the bound ADP molecule shown in blue. Amino acid residues interacting directly with the ligand are shown in stick representations. The 2mFO-DFC electron density map is shown in blue within a radius of 1.5 Å of any ligand atom and is contoured at 1σ. Electron density for the surrounding residues was omitted for clarity. Hydrogen bonds are shown as dotted black lines.

Figure S7
Interaction plots of (a) AMP and (b) ADP bound to adGlnK generated with program Ligplot+ (Laskowski & Swindells, 2011). Hydrogen bonds are shown as dotted green lines. Residues, which participate in hydrophobic interactions with the ligand, are highlighted by red circle segments.

Figure S8
Comparison of the T-loop conformations in adenylylated GlnK and in uridylylated GlnB.
(a) T-loop conformation in those three monomers present in the asymmetric unit of adGlnK in which adenylylated Tyr51 could be modelled. (b) T-loop conformation in those three monomers in adGlnK in which the adenylylated Tyr51 could not be modelled. In two of these three monomers, fortuitously bound nucleotides, i.e. either AMP or ADP, could be observed. (c) T-loop conformation in uridylylated GlnB with a bound ATP molecule (PDB code 5l9n, (Palanca & Rubio, 2017)).