PDBsum entry 1g8x

Structural protein

PDB id: 1g8x

Contents

Protein chains

1009 a.a. *

Ligands

ADP ×2

Metals

_MG ×2

Waters ×14

* Residue conservation analysis

PDB id:

1g8x

Name:

Structural protein

Title:

Structure of a genetically engineered molecular motor

Structure:

Myosin ii heavy chain fused to alpha-actinin 3. Chain: a, b. Fragment: myosin ii heavy chain, motor domain residues 1-761, and alpha-actinin 3, repeats 1 and 2 residues 765-1002. Engineered: yes. Mutation: yes

Source:

Dictyostelium discoideum. Organism_taxid: 44689. Expressed in: dictyostelium discoideum. Expression_system_taxid: 44689.

Resolution:

2.80Å R-factor: 0.232 R-free: 0.290

Authors:

W.Kliche,S.Fujita-Becker,M.Kollmar,D.J.Manstein,F.J.Kull

Key ref:

W.Kliche et al. (2001). Structure of a genetically engineered molecular motor. EMBO J, 20, 40-46. PubMed id: 11226153 DOI: 10.1093/emboj/20.1.40

Date:

21-Nov-00 Release date: 17-Jan-01

Protein chains

P05095  (ACTNA_DICDI) -  Alpha-actinin A from Dictyostelium discoideum

Seq: 861 a.a.

Struc: 1009 a.a.*

Protein chains

P08799  (MYS2_DICDI) -  Myosin-2 heavy chain from Dictyostelium discoideum

Seq: 2116 a.a.

Struc: 1009 a.a.*

* PDB and UniProt seqs differ at 466 residue positions (black crosses)

DOI no: 10.1093/emboj/20.1.40

EMBO J 20:40-46 (2001)

PubMed id: 11226153

Structure of a genetically engineered molecular motor.

W.Kliche, S.Fujita-Becker, M.Kollmar, D.J.Manstein, F.J.Kull.

ABSTRACT

Molecular motors move unidirectionally along polymer tracks, producing movement and force in an ATP-dependent fashion. They achieve this by amplifying small conformational changes in the nucleotide-binding region into force-generating movements of larger protein domains. We present the 2.8 A resolution crystal structure of an artificial actin-based motor. By combining the catalytic domain of myosin II with a 130 A conformational amplifier consisting of repeats 1 and 2 of alpha-actinin, we demonstrate that it is possible to genetically engineer single-polypeptide molecular motors with precisely defined lever arm lengths and specific motile properties. Furthermore, our structure shows the consequences of mutating a conserved salt bridge in the nucleotide-binding region. Disruption of this salt bridge, which is known to severely inhibit ATP hydrolysis activity, appears to interfere with formation of myosin's catalytically active 'closed' conformation. Finally, we describe the structure of alpha-actinin repeats 1 and 2 as being composed of two rigid, triple-helical bundles linked by an uninterrupted alpha-helix. This fold is very similar to the previously described structures of alpha-actinin repeats 2 and 3, and alpha-spectrin repeats 16 and 17.

Selected figure(s)

Figure 1.
Figure 1 Structure of M761-2R-R238E. Although two molecules are present in the crystallographic asymmetric unit, only one is shown here. The two molecules are essentially identical throughout the myosin motor domain (residues 2 -761). However, upon leaving the converter domain, the lever arms assume slightly different orientations and deviate at the ends by 19.4 Å. (A) A complete molecule spanning amino acids 2 -1010 is shown. No electron density was observed for five residues at the N-terminus, the loop region 205 -208 and one residue at the C-terminus. The N-terminal domain (2 -200) is shown in green; 50 kDa domain in red (201 -613); C-terminal and converter domain in blue (614 -761); linker region in orange (762 -764); -actinin lever arm in yellow (765 -1003); and seven histidines from the His[8] purification tag in gray (1004 -1010). The linker region is composed of three residues (Leu-Gly-Arg) introduced during cloning. The observed lever arm is 140 Å long (measured from C of 761 to C of 1010). Each -actinin repeat contributes 65 Å, and the histidine purification tag another 10 Å. Helices 1 -3 make up the first -actinin repeat, and 4 -6 the second. The arrowhead indicates the -helical region linking the two repeats. The disruptive kink in helix 2 is caused by the presence of two adjacent proline residues (see Figure 4A). (B) Detailed view of the linker region joining the myosin converter domain to helix 1 of -actinin. The view is rotated 180° around a vertical axis from that in (A).

Figure 4.
Figure 4 The structure of -actinin repeats 1 and 2. (A) An -carbon chain trace of the six helices making up repeats 1 (helices labeled 1 -3) and 2 (helices labeled 4 -6) is shown in yellow. The 17 hydrophobic aromatic amino acid residues stabilizing the triple-helical packing are shown in green (seven tyrosines, six phenylalanines and four tryptophans). Two adjacent proline residues are shown in red, which cause a kink but not a break in -helix 2 of repeat 1. The uninterrupted -helix linking repeats 1 and 2 is shown in orange. (B) Detailed view of the linker region, highlighting the stabilizing hydrophobic and hydrogen bonding interactions. Colors and orientation are identical to those in (A). Side chains are shown as ball-and-stick models, with the exception of Asp796 and Ser797, in which only the -carbon atoms involved in hydrophobic contacts are shown for clarity. The salt bridge between Arg880 and Glu877, and the hydrogen bond between Arg880 and the carbonyl oxygen of Leu956 (also shown as a ball-and-stick model), are shown as dashed lines.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2001, 20, 40-46) copyright 2001.

Figures were selected by an automated process.