On the potential alternate binding change mechanism in a dimeric structure of Pyruvate Phosphate Dikinase

The pyruvate phosphate dikinase (PPDK) reaction mechanism is characterized by a distinct spatial separation of reaction centers and large conformational changes involving an opening-closing motion of the nucleotide-binding domain (NBD) and a swiveling motion of the central domain (CD). However, why PPDK is active only in a dimeric form and to what extent an alternate binding change mechanism could underlie this fact has remained elusive. We performed unbiased molecular dynamics simulations, configurational free energy computations, and rigidity analysis to address this question. Our results support the hypothesis that PPDK dimerization influences the opening-closing motion of the NBDs, and that this influence is mediated via the CDs of both chains. Such an influence would be a prerequisite for an alternate binding change mechanism to occur. To the best of our knowledge, this is the first time that a possible explanation has been suggested as to why only dimeric PPDK is active.


Crystal packing environments of the NBDs in PDB ID 5JVJ
The PDBePISA tool 1 reveals six symmetry-related interfaces involving the NBD (Supplementary Table S2, interfaces labeled in italics), forming two different crystal packing environments for the NBDs (Supplementary Table S4 Table S4, labeled in bold). While this analysis is qualitative only, it suggests that the crystal packing contributes favorably to, if not fosters, the occurrence of conformationally different states of the NBDs in the asymmetric unit. Notable support for the influence of crystal packing on the conformational state of that PPDK structure arises from the fact that for chain B with a closed and likely adenine nucleotide-bound NBD, the CD is located close to the PBD, rather than the NBD (Fig 1b) 2 . This arrangement is unexpected in view of the enzyme mechanism 2 and leads to an energetically unfavorable conformational state of that chain (see Fig. 6 in ref. 2 ). Figure          The results from the present study suggest furthermore that PPDK dimerization influences the opening-closing motion of an NBD mediated via the CDs of both chains. Mutual increases in structural stability due to dimerization as revealed by CNA are indicated by orange dashed lines. Our findings lend support to the hypothesis of an alternate binding change mechanism in the PPDK dimer: When the NBD of chain A is closing (1), the CD of chain A swivels towards the NBD (2), the CD of chain B swivels towards the PBD (3), and the NBD of chain B is opening (4) (illustrated by black arrows) and vice versa (illustrated by gray arrows).

Supplementary Figures
Figure S10: Proposed mechanism of dimeric PPDK. The proposed mechanism involves the following steps: (IIC→IA) Transfer of a phosphoryl group from ATP to H456 of the CD of chain B. (IB) ATP binding to the NBD of chain A leads to the closing of this NBD, which is coupled with the CD motion of chain A swiveling from the PBD to the NBD, and the CD motion of chain B swiveling from the NBD to the PBD; the latter CD transports the phosphoryl group from the NBD to the PBD of chain B. (IC→IIA) The phosphoryl group bound to H456 of the CD of chain B is transferred to pyruvate at the PBD of chain B to generate PEP. Steps IIA-C are identical to IA-C but now the functional roles of chains A and B are exchanged. Between IIC and IA as well as IC and IIA, reactions (1) and (2) mentioned in the main text take place.  [a] The dimer interface of PPDK is shown in bold; interfaces involving the NBD are shown in italics.

Supplementary Tables
[b] Number of interface residues.
[d] Solvation free energy gain upon formation of the interface, in kcal mol -1 .
[e] Number of hydrogen bonds.
[f] Number of salt bridges.
[g] Complexation significance score.  [a] Interface residues, predicted by PDBePISA. [b] Residues forming hydrogen bonds and salt bridges are labeled in bold and blue, respectively.
[c] Accessible surface area, in Å 2 , predicted by PDBePISA. [d] Buried surface area, in Å 2 , with each vertical bar corresponding to 10% of total solventaccessible surface area buried, predicted by PDBePISA.
[f] Degree of conservation, from a multiple sequence alignment obtained with MAFFT 4 of 1000 PPDK sequences identified by BLASTp 5 in the NCBI-NR database of non-redundant protein sequences 6 .