Derek N. Woolfson

The design of coiled-coil structures and assemblies

Protein design allows sequence-to-structure relationships in proteins to be examined and, potentially, new protein structures and functions to be made to order. To succeed, however, the protein-design process requires reliable rules that link protein sequence to structure?function. Although our present understanding of coiled-coil folding and assembly is not complete, through numerous bioinformatics and experimental studies there are now sufficient rules to allow confident design attempts of naturally observed and even novel coiled-coil motifs. This review summarizes the current design rules for coiled coils, and describes some of the key successful coiled-coil designs that have been created to date. The designs range from those for relatively straightforward, naturally observed structures-including parallel and antiparallel dimers, trimers and tetramers, all of which have been made as homomers and heteromers-to more exotic structures that expand the repertoire of Natures coiled-coil structures. Examples in the second bracket include a probe that binds a cancer-associated coiled-coil protein; a tetramer with a right-handed supercoil; sticky-ended coiled coils that self-assemble to form fibers; coiled coils that switch conformational state; a three-component two-stranded coiled coil; and an antiparallel dimer that directs fragment complementation of larger proteins. Some of the more recent examples show an important development in the field; namely, new designs are being created with function as well as structure in mind. This will remain one of the key challenges in coiled-coil design in the next few years. Other challenges that lie ahead include the need to discover more rules for coiled-coil prediction and design, and to implement these in prediction and design algorithms. The considerable success of coiled-coil design so far bodes well for this, however. It is likely that these challenges will be met and surpassed.

Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)- domain co-chaperones

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co‐chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR‐domain co‐chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub‐micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co‐chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP‐binding site in the N‐terminal domain of Hsp90. These results reveal an important role for TPR‐domain co‐chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP‐dependent step in Hsp90‐mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client‐protein release.

Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37

In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a “scaffold,” recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50 cdc37 , whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50 cdc37 , like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a “relaxed” ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50 cdc37 binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50 cdc37 is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50 cdc37 forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo.

Core-directed protein design.

For various reasons, it seems sensible to redesign or design proteins from the inside out. Past approaches in this field have involved iterations of mutagenesis and characterisation to evolve designs. Increasingly, combinatorial approaches are being taken to select fit sequences from libraries of variant proteins. In particular, in silico methods have been used to good effect. More recently, experimental methods have been developed and improved. We are now in a position to redesign stability and function into natural protein frameworks confidently and to attempt de novo designs for more ambitious targets.

Coiled-coil assembly by peptides with non-heptad sequence motifs

BACKGROUNDnThe seven-residue heptad repeat is the accepted hallmark of coiled coils. In extended filamentous proteins, however, contiguous patterns of heptads are often disrupted by skips and stammers. The structural consequences and roles of these digressions are not understood.nnnRESULTSnIn a cytoskeleton protein from Giardia lamblia, heptads flank eleven-residue units (hendecads) to give a 7-11-7 motif that dominates the sequence. Synthetic peptides made to the consensus sequence of this motif fold in solution to fully helical, parallel dimers. Both the sequence pattern and these experimental data are consistent with the coiled-coil model. We note that breaks in other extended coiled coils can also be reconciled by hendecad insertions.nnnCONCLUSIONSnThe heptad paradigm for the coiled coil must be expanded to include hendecads. As different combinations of heptads and hendecads will give different overall sequence motifs, we propose that these provide a mechanism to promote cognate protein pairings during the folding of extended coiled coils in the cell.

Mini-proteins Trp the light fantastic

A new 20-residue peptide represents the smallest example to date of cooperatively folded tertiary structure. This achievement provides a new tool for elucidating protein conformational preferences. The mini-protein should serve as a fruitful platform for protein design.

Open‐and‐shut cases in coiled‐coil assembly: α‐sheets and α‐cylinders

The coiled coil is a ubiquitous protein‐folding motif. It generally is accepted that coiled coils are characterized by sequence patterns known as heptad repeats. Such patterns direct the formation and assembly of amphipathic α‐helices, the hydrophobic faces of which interface in a specific manner first proposed by Crick and termed “knobs‐into‐holes packing”. We developed software, socket, to recognize this packing in protein structures. As expected, in a trawl of the protein data bank, we found examples of canonical coiled coils with a single contiguous heptad repeat. In addition, we identified structures with multiple, overlapping heptad repeats. This observation extends Cricks original postulate: Multiple, offset heptad repeats help explain assemblies with more than two helices. Indeed, we have found that the sequence offset of the multiple heptad repeats is related to the coiled‐coil oligomer state. Here we focus on one particular sequence motif in which two heptad repeats are offset by two residues. This offset sets up two hydrophobic faces separated by ≈150°–160° around the α‐helix. In turn, two different combinations of these faces are possible. Either similar or opposite faces can interface, which leads to open or closed multihelix assemblies. Accordingly, we refer to these two forms as α‐sheets and α‐cylinders. We illustrate these structures with our own predictions and by reference to natural variants on these designs that have recently come to light.

Chemical synthesis and cytotoxicity of dihydroxylated cyclopentenone analogues of neocarzinostatin chromophore.

Compounds containing the naphthoate moiety of Neocarzinostatin chromophore or 2-hydroxynaphthoate have been synthesized and evaluated for cytotoxic activity against a leukemia cell line and a small panel of human-tumor cell lines. Those compounds containing a cyclopentenone moiety were active, with the carbonyl group being essential for biological activity.