Missing from this picture was a structure of an SRP–SR complex to provide the molecular basis for the reciprocal activation of GTPase activity. Now, a group of researchers from the University of California, San Francisco, has determined the structure of the catalytic core of the SRP–SR complex in the thermophilic bacteria Thermus aquaticus with one molecule of GMPPCP, a nonhydrolyzable GTP analogue, bound in each subunit. The researchers used data gathered at ALS Beamline 8.3.1 and the molecular-replacement method to obtain the first high-resolution structure (1.9 Å) of a protein-targeting complex.

The SRP-mediated protein-targeting cycle. As a nascent protein emerges from the ribosome, its signal sequence (yellow) is recognized by the signal recognition particle (SRP), which subsequently associates with its membrane-associated receptor (SR). This critical step precedes the selective attachment of the ribosome–nascent chain complex to the translocon in the membrane.

A Seeing Eye for Proteins

Proteins are fundamental components of all living cells and include many substances, such as enzymes, hormones, and antibodies, that are necessary for the proper functioning of an organism. Following a recipe provided by DNA, ribosomes are the factories that manufacture proteins, spitting out link by link a chain that eventually folds in on itself and becomes the functioning protein. Where the ribosomes are, however, is not necessarily where the proteins will report for duty. In the case of proteins bound for membranes, a signal recognition particle (SRP) sticks to the partially completed protein and guides it, along with the ribosome, to the membrane, where the SRP joins with a matching SRP receptor (SR). The SRP–SR complex then releases the protein, which lodges at its new home in the membrane. The formation of the SRP–SR complex and its subsequent dissociation are key events in this process. Egea et al. have obtained the first high-resolution structure (1.9 Å) of an SRP–SR complex from a bacterium (the same type of complex exists and operates in most forms of life, including humans). Their structure provides the molecular basis for a model of protein-targeting to biological membranes in which the formation of the complex is coupled to its subsequent disassembly, thereby insuring that the targeting brings the protein to the membrane but does not remove it (the targeting is unidirectional).

The SRP and SR proteins are known to be closely related GTPases that belong to the SRP-GTPase family, and their biological role in protein targeting is conserved across all the kingdoms of life. Both SRP and SR proteins include G domains with additional insertion box domains and N domains. However, in contrast to most other members of the GTPase superfamily, activation of the SRP family GTPases is triggered by heterodimer formation between the two homologous GTPase domains in which the G domain and the amino terminal N domain of each are structurally and functionally coupled in an arrangement universally conserved between all SRP-GTPases.

In the structure determined by the UCSF group, the two partners form a quasi-twofold symmetrical heterodimer. The interaction surface between the two subunits is extensive (3200 Ų), and involves conserved residues from the N domains and G domains of both proteins. A conserved ALLEADV sequence in the N domain and a conserved loop in the insertion box domain (IBD) define the edges of the heterodimer interface. The highly cooperative formation of the complex aligns the two GTP molecules in a symmetrical, composite active site where the 3'OH of one GTP is hydrogen bonded to the γ-phosphate of the other. This unprecedented circle of twinned interactions is severed twice upon hydrolysis, leading to complex dissociation after cargo delivery. Biochemical analysis supports the importance of the extensive interaction surface and the role of the 3'OH groups for association, reciprocal activation, and catalysis.

Structure of the quasi-symmetrical heterodimer of the catalytic core of the SRP–SR complex with the two GMPPCPs twinned in the shared GTP-binding cavity formed at the interface of the complex. The SRP and SR subunits are colored in blue and green, respectively. Two different orientations are shown, parallel (left) and along the quasi-twofold axis displayed in magenta (right).

The overall quasi-twofold symmetry of the complex is beautifully reflected in the catalytic site with the twinning of the GTP substrates and the symmetry of protein–GTP interactions. The two IBD motifs rearrange and contribute six residues (three each from SRP and SR) of central importance in the composite active site. Four of these residues are brought into position to stabilize the transition state. The resolution of the x-ray data allows the identification of the two strictly conserved catalytic aspartate residues that position a nucleophilic attacking water proximal to the γ-phosphate of each GTP.

Stereo view of electron density showing the two twinned GTP analogues (GMPPCPs) in close contact in the composite catalytic site with the symmetrical hydrogen bonds between the 3'OH ribose of one GTP and the γ-phosphate of the other. The two strictly conserved catalytic aspartates positioning the two attacking waters (red spheres) are displayed together with the two magnesium ions (yellow spheres) involved in GTP coordination and binding.

In sum, the structure explains the most basic requirement for SRP-dependent unidirectional targeting, namely the coupling of the SRP–SR complex formation with its subsequent disassembly, and suggests a unique activation mechanism for the SRP family of GTPases. The extensive interactions, both from active-site residues and from the twinned substrate, explain why complex formation is GTP-dependent and why GTP hydrolysis leads to complex dissociation.

Research conducted by P.F. Egea, S. Shan, J. Napetschnig, D.F. Savage, P. Walter, and R.M. Stroud (University of California, San Francisco).

Research funding: National Institutes of Health, Herbert Boyer Fund, and Burroughs-Wellcome Fund. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: P.F. Egea, S. Shan, J. Napetschnig, D.F. Savage, P. Walter, and R.M. Stroud, "Substrate twinning activates the signal recognition particle and its receptor," Nature 427, 215 (2004).

ALSNews Vol. 244, August 25, 2004