Rational 'correction' of the amino-acid sequence of RbcX protein from the thermophilic cyanobacterium Thermosynechococcus elongatus dramatically improves crystallization

Abstract

RbcX is a dimeric protein found in cyanobacteria that assists in the assembly of the oligomeric RuBisCO complex. RbcX from the thermophile Thermosynechococcus elongatus (TeRbcX) contains an unusual Cys103 residue in its sequence and when expressed recombinantly the protein aggregates and cannot be crystallized. Site-directed mutagenesis of Cys103 to either Arg or Ala produced non-aggregating proteins that could be readily crystallized in several crystal forms. Synchrotron-radiation X-ray diffraction data were collected to 1.96 A resolution and formed the basis of crystal structure analysis of TeRbcX.

Figure 1

Sequence alignment of RbcX sequences from T. elongatus (Te; GenBank accession No. NP_682295), Synechococcus sp. PCC7002 (7002; BAA03077), Synechocystis sp. PCC6803 (6803; BAA10191), Synechococcus sp. PCC7942 (7942; ABB57565) and Anabaena sp. CA (A_CA, AAA63603). The sequences were aligned using ClustalX (Thompson et al., 1997 ▶) and visualized with ESPript. Residues with strict identity are shown as white characters on a red background, whereas those with high similarity are shown as red characters and framed. The Cys103 residue of TeRbcX and the corresponding conserved arginine residue are highlighted with yellow and blue backgrounds, respectively. Sequence identity/similarity (%) with respect to T. elongatus RbcX is shown for all the remaining sequences.

Figure 2

Native PAGE analysis of purified TeRbcX. Lane 1, wild-type TeRbcX purified in the absence of DTT; lane 2, wild-type TeRbcX purified in the presence of DTT; lane 3, C103R mutant protein purified in the absence of DTT; lane 4, C103A mutant protein purified in the absence of DTT.

Figure 3

Crystals of TeRbcX: (a) form A-1, (b) form A-2, (c) form R-1, (d) form R-2.

Figure 4

An X-ray diffraction pattern recorded from a form A-1 crystal of TeRbcX (oscillation range 1°). The edge of the detector (inset) corresponds to a resolution of 1.96 Å.