PDBsum entry 2ft8

Electron transport

PDB id: 2ft8

Contents

Protein chain

122 a.a.

Metals

CU1

Waters ×154

References listed in PDB file

Key reference

Title

Basic requirements for a metal-Binding site in a protein: the influence of loop shortening on the cupredoxin azurin.

Authors

C.Li, S.Yanagisawa, B.M.Martins, A.Messerschmidt, M.J.Banfield, C.Dennison.

Ref.

Proc Natl Acad Sci U S A, 2006, 103, 7258-7263. [DOI no: 10.1073/pnas.0600774103]

PubMed id

16651527

Abstract

The main active-site loop of the copper-binding protein azurin (a cupredoxin) has been shortened from C(112)TFPGH(117)SALM(121) to C(112)TPH(115)PFM(118) (the native loop from the cupredoxin amicyanin) and also to C(112)TPH(115)PM(117). The Cu(II) site structure is almost unaffected by shortening, as is that of the Cu(I) center at alkaline pH in the variant with the C(112)TPH(115)PM(117) loop sequence. Subtle spectroscopic differences due to alterations in the spin density distribution at the Cu(II) site can be attributed mainly to changes in the hydrogen-bonding pattern. Electron transfer is almost unaffected by the introduction of the C(112)TPH(115)PFM(118) loop, but removal of the Phe residue has a sizable effect on reactivity, probably because of diminished homodimer formation. At mildly acidic pH values, the His-115 ligand protonates and dissociates from the cuprous ion, an effect that has a dramatic influence on the reactivity of cupredoxins. These studies demonstrate that the amicyanin loop adopts a conformation identical to that found in the native protein when introduced into azurin, that a shorter than naturally occurring C-terminal active-site loop can support a functional T1 copper site, that CTPHPM is the minimal loop length required for binding this ubiquitous electron transfer center, and that the length and sequence of a metal-binding loop regulates a range of structural and functional features of the active site of a metalloprotein.

Figure 1.
Fig. 1. The structure of AZ, with the C-terminal ligand-containing loop and the copper ion shown in purple. The side chains of the Cys-112, His-117, and Met-121 ligands on this loop and also of His-46 are shown as stick models in purple. The backbone carbonyl oxygen of Gly-45, which provides the second weak axial interaction at the copper site, and the helical nature of the His-117-to-Met-121 sequence are omitted for clarity. The figure was prepared by using the program PYMOL (6).

Figure 4.
Fig. 4. Stereoview of active sites. (A) Overlay of the active sites of Cu(II) AZAMI (green), AZAMI-F (gray), AZ (purple), and AMI (yellow). The side chains of the coordinating residues and the amino acids on either side of the N-terminal His ligand are shown as stick models, copper atoms as spheres, and the backbone of the C-terminal ligand-containing loops as C^ traces. The residues are labeled as in AZ. (B) Overlay of the active sites of the Cu(I) form of AZAMI-F at pH 8 (slate) and pH 6 (cyan) is shown. The second conformation of the copper ion and His-115 at pH 6 are colored green. The figure was prepared by using the program PYMOL (6).

Secondary reference #1

Title

Crystal structure analysis of oxidized pseudomonas aeruginosa azurin at ph 5.5 and ph 9.0. A ph-Induced conformational transition involves a peptide bond flip.

Authors

H.Nar, A.Messerschmidt, R.Huber, M.Van de kamp, G.W.Canters.

Ref.

J Mol Biol, 1991, 221, 765-772.

PubMed id

1942029

Secondary reference #2

Title

Loop-Contraction mutagenesis of type 1 copper sites.

Authors

S.Yanagisawa, C.Dennison.

Ref.

J Am Chem Soc, 2004, 126, 15711-15719. [DOI no: 10.1021/ja047295r]

PubMed id

15571393

Secondary reference #3

Title

Crystal structure analysis and refinement at 2.15 a resolution of amicyanin, A type i blue copper protein, From thiobacillus versutus.

Authors

A.Romero, H.Nar, R.Huber, A.Messerschmidt, A.P.Kalverda, G.W.Canters, R.Durley, F.S.Mathews.

Ref.

J Mol Biol, 1994, 236, 1196-1211. [DOI no: 10.1016/0022-2836(94)90021-3]

PubMed id

8120896

Figure 3.
Figure 3. A section (z= 14/42) of the difference Patterson map compute at 3.5 A resolution. Two sites relaed by translation of z, ) = (2/3, l/3) g' Ives rse to identical position in the difference Patterson map which in turn explains the elative peak heights of 2 : I or the 2 Harker peaks.

Figure 8.
Fgure 8. C:rvstal acking of amicyanin from T. versrt&. The 3 independent molecules of amicyanin are arranged as

The above figures are reproduced from the cited reference with permission from Elsevier