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Now that the X-ray structures of hun- dreds of protein molecules have been determined, one might not expect to come upon anything fundamentally new. Indeed, similarity with familiar protein folds is one criterion by which cautious crystallographers validate their structures. Yet pectate lyase C (PelC), an enzyme secreted by the bac- terium Envinia chrysanthemi during its depredations upon plant tissue, pre- sents so elegantly simple and novel a fold that a wily struoturH biologist might have invented it. Yo~er et al.~ call the single structural domain of PelC a [5-helix (Fig. la). This fold is generated by coiling a ~-strand into a right-handed helix. It could also be described as a superhelix, since [5-strands are them- selves helices. Each turn (of seven in total) around the superhelix axis is completed by three short ~-strand seg- ments, separated by turns (two of the ~- arch type, the third a sharp '[l-breaker' turn2). The [5-strands of consecutive superhelical turns line up along the

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superhelix axis to form three parallel ~sheets. Thus, PelC approximates to a triangular prism whose vertices are marked by the turns or bends that con- nect adjacent ~-strands.

What is so unusual about this struc- ture? Parallel ~-sheets are quite com- mon; in fact most enzymes are con- structed about parallel cores. The difference is in the structure of the crossover connections - always right- handed 3 - between consecutive strands in any one sheet, in PelC, the crossover connections comprise the ~-strands and intervening turns of the other two sheets. The sidechaln moieties at alter- nate residues project from opposite sides of the sheet, one into the prism, the next toward the solvent (since [5- strands are helices with approximately two residues per turn). In classic paral- lel ~-sheet structures, such as triose

phosphate isomerase ~Pl) , the archetypal parallel [~-barrel 4, crossover connections are made by ~-helices (Fig. lb). These helices are amphipathic; hydrophobic residues extend from the surface of the helix in contact with the [5-strand, while hydrophilic residues are exposed to solvent on the opposite sur- face. Eight such [~/c~ units form a closed barrel, so that the sides of the p-strands facing the interior of the barrel are also buried. (PeIC could be made to form a topologically equivalent barrel by curl- ing the ~superhelix about a central axis parallel to the strands, such that the opposite ends meet, like a slinky spring with the two ends abutting.) While most parallel ~-sheet structures are not simi- lar to TPI (the doubly wound parallel structures, of which the nucleotide- binding fold is a prototype, form the largest family), all have helical crossover connections that cover one or both sides of the [~-sheet; both inner and outer surfaces are shielded from solvent. Characteristically, both the

(a) (b) (c)

Rgure 1 Ribbon (,x-helix) and arrow (~-strand) representations of parallel []-sheet protein structures. Strands of the sheet closest to the viewer are tinted dark grey: (a) PelC from E. chrysanthemi, adapted from Yoder et al.1; (b) triose phosphate isomerase, adapted from Jane Richardson's Protein Coloring Book 9, is in fact a ~-barrel - the tinted strands do not represent a discrete face. The crossover connections are formed by helices; (©) the alkaline protease from P. aeruginosa. The Ca 2. ions bound at the GGXGXD motif half-sites are shown as

circles. Three other Ca 2* sites are also marked Ix).

© 1993, Elsevier Science Publishers, (UK) 0968-0004/93/$06.00 313

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inward and outward-facing residues of parallel sheets (domains) are hydro- phobic. Before the structure of PelC was elucidated, there was no example of a parallel ~sheet (domain) with its outer surface exposed to solvent, unprotected by amphipathic helices. This fostered the vague suspicion that parallel sheets are inherently less stable than their antiparallei cousins, which typically form two-layer [l-sheet sandwiches in which the exterior sur- face of each sheet is populated by hydrophilic residues, while the alter- nate, inward-facing residues are hydro- phobic - the immunoglobulin fold being the most familiar example. Neverthe- less, PelC is stable at room tempera- ture, even though almost 60% of the residues, many of them carrying polar sidechains on the exterior surface of the ~superhelix, are exposed to solvent (the rest are covered by surface loops or the amino- and carboxy-terminal segments o[ the polypeptide chain).

The unusual stability of PelC might be attributed to a regular set of packing interactions between residues within the 13-superhelix. The ~sheets of PelC are only slightly twisted (twist refers to the angle between successive strands in the sheet; 10-200 is usual [or most sheets). Consequently, the sidechains of residues at corresponding positions in consecutive strands stack directly upon each other, in contrast to the partially staggered arrangement in more twisted sheets. One very remark- able stack comprises a ladder of six buried asparagine sldechains, hydrogen bonded -NH., to -C=O, that link con- secutive turns of the ~-superhelix at the inner juncture between two ~-sheets, The binding energy in a chain of spe- cilic and oriented hydrogen bonding partners might easily compensate for the loss of chain entropy in bringing them together. Similarly, aromatic and aliphatic amino acid sidechains that project from adjacent residues in neigh- boring strands are aligned in rigid columns. In addition, PeIC contains a weakly bound Ca 2" ion that is required for activity, and that may promote the stability of the fold.

The PelC family, which includes both secreted and extracellular pectate ly- ases and perhaps also a group of plant pollen and style proteins, is not alone in its possession of a ~superhelix. Baumann et al. 5 now report that the Zn2"-dependent alkaline protease (AP) of Pseudomonas aeruginosa also con- tains a separate, noncatalytic domain

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with a ~-superhelix motif. In this instance, the central six strands of the domain are coiled into a '~-roll' of two opposing sheets (Fig. lc). Like the sheets of PeIC, there is little twist to the AP roll, so that again the hydrophobic residues that project into the roll are stacked. Moreover, the AP roll is stabil- ized by Ca 2~. The loops connecting seven consecutive turns of the parallel ~-roll contain the consensus seq~Jence GGXGXDXUX (X is any amin~ acid and U is large and hydrophobic, ideally leucine). Each such turn forms one half- site of a hexa-coordinated Ca 2÷ ion that bridges adjacent turns of the [I-roll. The first six residues of the consensus sequence form the Ca2÷-binding site, and the leucine is packed in the interior of the [l-roll.

The fact that examples of [i-roll or [5- helix motifs have only recently been discovered would suggest that they are uncommon. Could they play some special biological role? Yoder etal. ~ and Baumann et al. 5 suggest that ~-rolls might facilitate the passage of PelC or AP through the inner and outer mem- branes of the Gram-negative bacteria in which they are synthesized. However, the two proteins are secreted by differ- ent mechanisms. Applicable to both systems is the notion that, in order to pass through the outer membrane, the protein must undergo a reversible unfolding transition, The ~-roll motif would seem Ideal for such a transform. atlon since it might refold easily. PeIC uses a sec-dependent pathway 6 to cross the inner membrane. Export through the outer membrane is effected by pro- tein products of the out genes. It is not clear, however, whether all proteins exported by the out pathway will be found to contain ~-superhelix domains. In contrast, AP is secreted in a one-step, signal sequence-independent process through both inner and outer mem- branes utilizing protein products of the apr gene dusted. The functionally diverse proteins that are transported by this and related mechanisms contain multiple tandem repeats of the GGXGXDXUX sequence and thus per- haps ~roll motifs similar to that found in AP. A transport mechanism for these molecules might involve unfolding of the ~-roll domain in the intracellular or periplasmic space, followed by refold- ing of the domain in a Ca2+-rich extra- cellular milieu. Such speculations are ultimately testable by mutagenesis.

How strange are ~rolls and helices? The wealth of protein structures in the

databank 8 represent only a small, and perhaps skewed fraction of the protein kingdom. We should not be surprised to discover new, but perfectly reason- able - if unexpected - folds. Indeed, ~- rolls break none of the established 'rules' of protein structure. The rela- tively untwisted sheets and the conse- quent stacking interactions among in- terior residues seen in PelC and AP may prove to be a common feature of such structures. Perhaps parallel ~-sheets are more stable than physical bio- chemists might have once believed.

Acknowledgements i thank Drs Yoder, Jurnak, McKay and

their colleagues for sharing their data before publication, and Dr McKay for the atomic coordinates used to gener- ate Fig. lc. Thanks also to D. DeCamp for suggesting the title and E. Goldsmith for critical reading of the manuscript.

References 1 Yoder, M. D., Keen, N. T. and Jumak, F. (1993)

Science 260, 1503-1507 2 Cohen, F. E. (1993) Science 260, 1444-1445 3 Richardson, J. (1981) Adv. Protein Chem. 34,

167-339 4 Brand6n, C. and Tooze, J. (1991) Introduction to

Protein Structure, Garland 5 Baumann, U., Wu, S., Raherty, K. M. and

McKay, D. B. EMBO J. (in press) 6 He, S. Y., Schoedel, C., Chatterjee, A. K.

and Collmer, A, J. (1991) Bacterto/. 173, 4310-4317

7 Duong, F., Lazdunskl, A., Carol, B. and Murgier, M. (1992) Gone 121, 47-54

8 Bernsteln, F. C, et al. (1977) J, Mol. Biol. 112, 535-542

9 Richardson, J. (1979) In The Protein Coloring Book, Little River Institute

STEPHEN R. SPRANG

Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harff Hines Bird, Dallas, TX 75235-9050, USA.

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