2R82

Pyruvate phosphate dikinase (PPDK) triple mutant R219E/E271R/S262D adapts a second conformational state

Summary for 2R82

• Entry DOI: 10.2210/pdb2r82/pdb
• Related: 1DIK 1KBL 1KC7
• Descriptor: Pyruvate, phosphate dikinase, SULFATE ION (2 entities in total)
• Functional Keywords: phosphotransferase, conformational transition, swiveling domain, remote active sites, atp-binding, kinase, magnesium, metal-binding, nucleotide-binding, phosphorylation, transferase
• Biological source: Clostridium symbiosum
• Total number of polymer chains: 1
• Total formula weight: 96991.49

Authors

Lim, K.,Read, R.J.,Chen, C.C.,Herzberg, O. (deposition date: 2007-09-10, release date: 2008-01-01, Last modification date: 2023-08-30)

Primary citation

Lim, K.,Read, R.J.,Chen, C.C.,Tempczyk, A.,Wei, M.,Ye, D.,Wu, C.,Dunaway-Mariano, D.,Herzberg, O.
Swiveling domain mechanism in pyruvate phosphate dikinase.
Biochemistry, 46:14845-14853, 2007

PubMed Abstract: Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of phosphoenolpyruvate (PEP), AMP, and Pi to pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a three-residue mutant protein (R219E/E271R/S262D) that introduces repulsion between the His and nucleotide-binding domains but preserves viable interactions with the PEP/pyruvate-binding domain. Accordingly, the mutant enzyme is competent in catalyzing the PEP/pyruvate half-reaction but the overall activity is abolished. The new structure confirms the swivel motion of the His domain. In addition, upon detachment from the His domain, the two nucleotide-binding subdomains undergo a hinge motion that opens the active-site cleft. A similar hinge motion is expected to accompany nucleotide binding (cleft closure) and release (cleft opening). A model of the coupled swivel and cleft opening motions was generated by interpolation between two end conformations, each with His455 positioned for phosphoryl group transfer from/to one of the substrates. The trajectory of the His domain avoids major clashes with the partner domains while preserving the association of the two linker segments.

PubMed: 18052212
DOI: 10.1021/bi701848w

Experimental method

X-RAY DIFFRACTION (3.6 Å)