Natural beta-sheet proteins use negative design to avoid edge-to-edge aggregation

Abstract

The fact that natural beta-sheet proteins are usually soluble but that fragments or designs of beta structure usually aggregate suggests that natural beta proteins must somehow be designed to avoid this problem. Regular beta-sheet edges are dangerous, because they are already in the right conformation to interact with any other beta strand they encounter. We surveyed edge strands in a large sample of all-beta proteins to tabulate features that could protect against further beta-sheet interactions. beta-barrels, of course, avoid edges altogether by continuous H-bonding around the barrel cylinder. Parallel beta-helix proteins protect their beta-sheet ends by covering them with loops of other structure. beta-propeller and single-sheet proteins use a combination of beta-bulges, prolines, strategically placed charges, very short edge strands, and loop coverage. beta-sandwich proteins favor placing an inward-pointing charged side chain on one of the edge strands where it would be buried by dimerization; they also use bulges, prolines, and other mechanisms. One recent beta-hairpin design has a constrained twist too great for accommodation into a larger beta-sheet, whereas some beta-sheet edges are protected by the bend and reverse twist produced by an Lbeta glycine. All free edge strands were seen to be protected, usually by several redundant mechanisms. In contrast, edge strands that natively form beta H-bonded dimers or rings have long, regular stretches without such protection. These results are relevant to understanding how proteins may assemble into beta-sheet amyloid fibers, and they are especially applicable to the de novo design of beta structure. Many edge-protection strategies used by natural proteins are beyond our current abilities to constrain by design, but one possibility stands out as especially useful: a single charged side chain near the middle of what would ordinarily be the hydrophobic side of the edge beta strand. This minimal negative-design strategy changes only one residue, requires no backbone distortion, and is easy to design. The accompanying paper [Wang, W. & Hecht, M. H. (2002) Proc. Natl. Acad. Sci. USA 99, 2760-2765] makes use of the inward-pointing charge strategy with great success, turning highly aggregated beta-sandwich designs into soluble monomers.

Figure 1

End view of the left-handed, three-sheet β-helix of the 1QRE archaeal carbonic anhydrase (27). β strands are peach-colored arrows, with the three edge strands emphasized in darker orange. As is true for almost all β-helix ends, those edge strands are covered by a substantial loop, in this case containing an α-helix (in gold).

Figure 2

View down the approximate 5-fold axis of the β-propeller five structure of the 1TL2 tachylectin (28). β strands are lilac arrows, with edge strands of the five propeller “blades” in darker purple. For the edge strand on the right, the features preventing further β-sheet interactions are shown: a charged Lys, a Gly β bulge at the end of the strand, a nearby partially covering loop (pink), and a colinear helix (pink) that blocks the first β residue.

Figure 3

(a–d) A set of equivalent, paired sheet edges from four different β-sandwich protein families having the lectin/glucanase fold. Edge β strands are shown as sea green arrows, β bulges in hot pink, and inward-pointing charged side chains with red or blue balls for their O or N atoms. All examples have at least one bulge and at least one inward-pointing charge, although the positioning and the overall shapes vary. From PDB files 1NLS (29), 1SLT (30), 1SAC (31), and 1KIT (32).

Figure 4

A β-sheet edge strand (green arrow) that uses an Lβ Gly (labeled) to produce a large bend and a negative backbone twist for that end of the strand. The β H-bonding of that strand is completely regular, but the “pleat” is convex outward for three residues in a row (Lys, Gly, Gln). From the 1IGD single-sheet structure of an IGG-binding domain of Streptococcal protein G (33).

Figure 5

Edge β strands and dimer contacts for the 1TTA transthyretin β-sandwich structure (34). (a) Edge strands (green arrows) in the monomer, with a protective charged Arg and two loops (main chain in pink) only at the extreme ends, and long central stretches of completely regular β strand, especially on the longer strand at left. (b) That longer edge strand in its dimer contact, with six intersubunit β H-bonds (view rotated 90° from a). The shorter edge strand contact (behind; not shown) fits less well, with one H-bond at each end and bridging waters in the center.

Figure 6

A comparison of the high-twist, designed 1HS0 TrpZip4 β-hairpin (20) with the 46–61 β-hairpin from the intact 1IGD protein G structure (33). (a) Model 1 of the TrpZip4 NMR structure, with β-strand ribbons to emphasize its high twist and bend, and with all-atom contacts shown as color-coded dots (14). The Trp–Trp pairs form good contacts (green dots) on the convex side of the β-hairpin. (b) The similar but low-twist β-hairpin from protein G, with a Trp–Trp pair modeled and optimized as well as possible without backbone motion. The red spikes of their all-atom contacts show their physically impossible interpenetration.