PDBsum entry 2vii

Transcription

PDB id

2vii

Contents

			Protein chain

					247 a.a.

			Ligands

					AMP

			Metals

					_MG

			Waters ×107

References listed in PDB file

Key reference

Title

Trapping of a transcription complex using a new nucleotide analogue: AMP aluminium fluoride.

Authors

N.Joly, M.Rappas, M.Buck, X.Zhang.

Ref.

J Mol Biol, 2008, 375, 1206-1211. [DOI no: 10.1016/j.jmb.2007.11.050]

PubMed id

18082766

Abstract

Mechanochemical proteins rely on ATP hydrolysis to establish the different functional states required for their biological output. Studying the transient functional intermediate states these proteins adopt as they progress through the ATP hydrolysis cycle is key to understanding the molecular basis of their mechanism. Many of these intermediates have been successfully 'trapped' and functionally characterised using ATP analogues. Here, we present a new nucleotide analogue, AMP-AlF(x), which traps PspF, a bacterial enhancer binding protein, in a stable complex with the sigma(54)-RNA polymerase holoenzyme. The crystal structure of AMP-AlF(x)*PspF(1-275) provides new information on protein-nucleotide interactions and suggests that the beta and gamma phosphates are more important than the alpha phosphate in terms of sensing nucleotide bound states. In addition, functional data obtained with AMP-AlF(x) establish distinct roles for the conserved catalytic AAA(+) (ATPases associated with various cellular activities) residues, suggesting that AMP-AlF(x) is a powerful new tool to study AAA(+) protein family members and, more generally, Walker motif ATPases.



	Figure 1. Fig. 1. Identification and characterisation of new stable PspF[1–275] WT•σ^54 or Eσ^54 complexes in the presence of AMP–AlF[x]. (a) Native gel showing the complexes formed by PspF[1–275] WT (5 μM) with or without σ^54 (1 μM) and with or without RNA polymerase (0.15 μM) in the presence of AlF[x] reactant [AlC[3] (0.4 mM) + NaF (5 mM)] and different nucleotides (4 mM, as indicated). The sample was loaded onto native 4.5% PAGE and proteins were detected by Coomassie blue staining. ANP indicates AMP or ADP. (b) Gel filtration profiles of samples containing PspF[1–275] WT (64 μM) with or without σ^54 (30 μM) and with or without AMP–AlF[x] or ADP–AlF[x] (as indicated) chromatographed through a Superdex 200 column (10 mm × 300 mm, 24 ml, GE Healthcare) at 4 °C. The scale bars give the scale of the ordinate axis; absorption units (AU) correspond to an A[280 nm] of 1.		Figure 2. Fig. 2. Final 2F[o] − F[c] and omit difference F[o] − F[c] electron density maps of the nucleotide-binding pocket of PspF[1–275]. (a) Final 2F[o] − F[c] map of the Mg-AMP-PspF[1–275] structure at 2.85 Å resolution contoured at 1σ. The neighbouring subunit is coloured magenta. Important intramolecular catalytic residues are highlighted and important intermolecular catalytic residues offered by the neighbouring subunit are denoted ‘t’ for ‘trans’. Note how the extra electron density encircled in green dashed lines occupies the position of the γ phosphate in ATP-bound structures of PspF[1–275] and is connected to the electron density of the AMP moiety. (b) Omit difference F[o] − F[c] map of the Mg-AMP-PspF[1–275] structure contoured at 3σ. The PspF[1–275]-ATP-bound structure was superimposed onto the P loop of the PspF[1–275]-Mg-AMP-bound structure and the result is displayed in this figure. Note that ATP fits convincingly into the difference map. Further note how the γ phosphate of ATP fits into the extra density encircled in (a). Data were collected at ESRF beamline ID-29. Refinement of the structure was performed as described^14 and the nucleotide and the Mg ion were refined with unit occupancy. Map inspection, model building and water molecule picking were done using Coot.^18 The average temperature factors in this structure are 30 for the protein, 60 for the AMP and 57 for the Mg. For analysis, all the liganded (ATP: 2C96; ADP: 2C98; AMPPNP: 2C99; Mg-ATP-PspF[1–275]R227A: 2C9C) and unliganded (Apo: 2BJW) PspF structures were aligned onto their P loops (residues 35 to 43). All figures were prepared using Pymol (Delano, W. L. (2002). The PyMOL Molecular Graphics System on the World Wide Web, http://www.pymol.org).

PROCHECK

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