From rhomboid function to structure and back again

It is said that “a picture is worth a thousand words,” a snapshot or an image of a complex reality, but it can never tell the whole story. Likewise, protein crystal structures divulge important molecular features, but a comprehensive understanding requires an arsenal of techniques and expertise from a variety of fields. The interplay between and importance of structural and functional studies are nicely illustrated by recent reports on the membrane-embedded protease rhomboid. For several years, molecular and biochemical findings provided strong evidence that rhomboids were a new type of serine protease with an active site within the boundaries of the lipid bilayer (1). In the past several months, studies of five crystal structures (2–6) have confirmed this theory but have offered conflicting ideas regarding substrate access to the active site. In this issue of PNAS, Baker et al. (7) bring us full circle, presenting compelling functional support for one of these hypotheses.

Rhomboids are found in virtually all organisms and were first discovered as proteases in 2001 (8). Evidence for proteolytic function included conserved serine and histidine residues that were essential for activity in cells, sensitivity to certain serine protease inhibitors, and a cleavage site within the transmembrane (TM) region of the substrate. Bacterial expression and purification (9, 10) further demonstrated that rhomboids work alone, without the need for other protein factors. The recent crystal structures (all of bacterial rhomboid orthologs) have confirmed the six-TM helix topology of the protein (Fig. 1 a) and removed any doubt about their specific identity as serine proteases: the key Ser-201 and His-254 residues, located on TM helices 4 and 6, respectively, are indeed adjacent to each other and oriented in essentially the same way as found in classical soluble serine proteases. Two unusual features of the structure include a …

*To whom correspondence should be addressed. E-mail: mwolfe{at}rics.bwh.harvard.edu