Labile assembly of a tardigrade protein induces biostasis.

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self-assembly of labile CAHS gels.

Computer simulations of the growth of synthetic peptide fibres.

We present a coarse-grained computer model designed to study the growth of fibres in a synthetic self-assembling peptide system. The system consists of two 28 residue α-helical sequences, denoted AB and CD, in which the interactions between the half peptides, A, B, C and D, may be tuned individually to promote different types of growth behaviour. In the model, AB and CD are represented by double ended rods, with interaction sites distributed along their lengths. Monte Carlo simulations are performed to follow fibre growth. It is found that lateral and longitudinal growth of the fibre are governed by different mechanisms--the former is diffusion limited with a very small activation energy for the addition of units, whereas the latter occurs via a process of secondary nucleation at the fibre ends. As a result, longitudinal growth generally proceeds more slowly than lateral growth. Furthermore, it is shown that the aspect ratio of the growing fibre may be controlled by adjusting the temperature and the relative strengths of the interactions. The predictions of the model are discussed in the context of published data from real peptide systems.

Amino acid pairing preferences in parallel beta-sheets in proteins.

Statistical approaches have been applied to examine amino acid pairing preferences within parallel beta-sheets. The main chain hydrogen bonding pattern in parallel beta-sheets means that, for each residue pair, only one of the residues is involved in main chain hydrogen bonding with the strand containing the partner residue. We call this the hydrogen bonded (HB) residue and the partner residue the non-hydrogen bonded (nHB) residue, and differentiate between the favorability of a pair and that of its reverse pair, e.g. Asn(HB)-Thr(nHB)versus Thr(HB)-Asn(nHB). Significantly (p < or = 0.000001) favoured pairings were rationalised using stereochemical arguments. For instance, Asn(HB)-Thr(nHB) and Arg(HB)-Thr(nHB) were favoured pairs, where the residues adopted favoured chi1 rotamer positions that allowed side-chain interactions to occur. In contrast, Thr(HB)-Asn(nHB) and Thr(HB)-Arg(nHB) were not significantly favoured, and could only form side-chain interactions if the residues involved adopted less favourable chi1 conformations. The favourability of hydrophobic pairs e.g. Ile(HB)-Ile(nHB), Val(HB)-Val(nHB) and Leu(HB)-Ile(nHB) was explained by the residues adopting their most preferred chi1 and chi2 conformations, which enabled them to form nested arrangements. Cysteine-cysteine pairs are significantly favoured, although these do not form intrasheet disulphide bridges. Interactions between positively and negatively charged residues were asymmetrically preferred: those with the negatively charged residue at the HB position were more favoured. This trend was accounted for by the presence of general electrostatic interactions, which, based on analysis of distances between charged atoms, were likely to be stronger when the negatively charged residue is the HB partner. The Arg(HB)-Asp(nHB) interaction was an exception to this trend and its favorability was rationalised by the formation of specific side-chain interactions. This research provides rules that could be applied to protein structure prediction, comparative modelling and protein engineering and design. The methods used to analyse the pairing preferences are automated and detailed results are available (http://www.rubic.rdg.ac.uk/betapairprefsparallel/).

Guidelines for the assembly of novel coiled-coil structures: alpha-sheets and alpha-cylinders.

The coiled coil is a ubiquitous motif that guides many different protein-protein interactions. The accepted hallmark of coiled coils is a seven-residue (heptad) sequence repeat. The positions of this repeat are labelled a-b-c-d-e-f-g, with residues at a and d tending to be hydrophobic. Such sequences form amphipathic alpha-helices, which assemble into helical bundles via knobs-into-holes interdigitation of residues from neighbouring helices. We wrote an algorithm, SOCKET, to identify this packing in protein structures, and used this to gather a database of coiled-coil structures from the Protein Data Bank. Surprisingly, in addition to commonly accepted structures with a single, contiguous heptad repeat, we identified sequences with multiple, offset heptad repeats. These 'new' sequence patterns help to explain oligomer-state specification in coiled coils. Here we focus on the structural consequences for sequences with two heptad repeats offset by two residues, i.e. a/f'-b/g'-c/a'-d/b'-e/c'-f/d'-g/e'. This sets up two hydrophobic seams on opposite sides of the helix formed. We describe how such helices may combine to bury these hydrophobic surfaces in two different ways and form two distinct structures: open 'alpha-sheets' and closed 'alpha-cylinders'. We highlight these with descriptions of natural structures and outline possibilities for protein design.

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.

Open-and-shut cases in coiled-coil assembly: alpha-sheets and alpha-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 alpha-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 Crick's 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 approximately 150 degrees -160 degrees around the alpha-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 alpha-sheets and alpha-cylinders. We illustrate these structures with our own predictions and by reference to natural variants on these designs that have recently come to light.

Biophysical analysis of natural variants of the multimerization region of Epstein-Barr virus lytic-switch protein BZLF1.

BZLF1 plays a key role in the induction of Epstein-Barr virus (EBV) replication. On the basis of limited sequence homology and mutagenesis experiments, BZLF1 has been described as a member of the bZip family of transcription factors, but this prospect has not been rigorously tested to date. Here, we present biophysical analysis of the multimerization domain of BZLF1, from three natural variants of EBV, and demonstrate for the first time that the region between amino acids 196 and 227 is sufficient to direct folding as a coiled-coil dimer in vitro.

Socket: a program for identifying and analysing coiled-coil motifs within protein structures.

The coiled coil is arguably the simplest protein-structure motif and probably the most ubiquitous facilitator of protein-protein interactions. Coiled coils comprise two or more alpha-helices that wind around each other to form "supercoils". The hallmark of most coiled coils is a regular sequence pattern known as the heptad repeat. Despite this apparent simplicity and relatedness at the sequence level, coiled coils display a considerable degree of structural diversity: the helices may be arranged parallel or anti-parallel and may form a variety of oligomer states. To aid studies of coiled coils, we developed SOCKET, a computer program to identify these motifs automatically in protein structures. We used SOCKET to gather a set of unambiguous coiled-coil structures from the RCSB Protein Data Bank. Rather than searching for sequence features, the algorithm recognises the characteristic knobs-into-holes side-chain packing of coiled coils; this proved to be straightforward to implement and was able to distinguish coiled coils from the great majority of helix-helix packing arrangements observed in globular domains. SOCKET unambiguously defines coiled-coil helix boundaries, oligomerisation states and helix orientations, and also assigns heptad registers. Structures retrieved from the Protein Data Bank included parallel and anti-parallel variants of two, three and four-stranded coiled coils, one example of a parallel pentamer and a small number of structures that extend the classical description of a coiled coil. We anticipate that our structural database and the associated sequence data that we have gathered will be of use in identifying principles for coiled-coil assembly, prediction and design. To illustrate this we give examples of sequence and structural analyses of the structures that are possible using the new data bases, and we present amino acid profiles for the heptad repeats of different motifs.

Sticky-end assembly of a designed peptide fiber provides insight into protein fibrillogenesis.

Coiled-coil motifs provide simple systems for studying molecular self-assembly. We designed two 28-residue peptides to assemble into an extended coiled-coil fiber. Complementary interactions in the core and flanking ion-pairs were used to direct staggered heterodimers. These had "sticky-ends" to promote the formation of long fibers. For comparison, we also synthesized a permuted version of one peptide to associate with the other peptide and form canonical heterodimers with "blunt-ends" that could not associate longitudinally. The assembly of both pairs was monitored in solution using circular dichroism spectroscopy. In each case, mixing the peptides led to increased and concentration-dependent circular dichroism signals at 222 nm, consistent with the desired alpha-helical structures. For the designed fiber-producing peptide mixture, we also observed a linear dichroism effect during flow orientation, indicative of the presence of long fibrous structures. X-ray fiber diffraction of partially aligned samples gave patterns indicative of coiled-coil structure. Furthermore, we used electron microscopy to visualize fiber formation directly. Interestingly, the fibers observed were at least several hundred micrometers long and 20 times thicker than expected for the dimeric coiled-coil design. This additional thickness implied lateral association of the designed structures. We propose that complementary features present in repeating structures of the type we describe promote lateral assembly, and that a similar mechanism may underlie fibrillogenesis in certain natural systems.

A ligand-reversible dimerization system for controlling protein-protein interactions.

Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains are fused to a signaling domain of interest, allowing crosslinking to be initiated by addition of a bivalent FKBP ligand. In the course of protein engineering studies on human FKBP, we discovered that a single point mutation in the ligand-binding site (Phe-36 --> Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analytical ultracentrifugation, and x-ray crystallographic studies show that the mutant (F(M)) forms discrete homodimers with micromolar affinity that can be completely dissociated within minutes by addition of monomeric synthetic ligands. These unexpected properties form the basis for a "reverse dimerization" regulatory system involving F(M) fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have used this strategy to rapidly and reversibly aggregate fusion proteins in different cellular compartments, and to provide an off switch for transcription. Reiterated F(M) domains should be generally useful as conditional aggregation domains (CADs) to control intracellular events where rapid, reversible dissolution of interactions is required. Our results also suggest that dimerization is a latent property of the FKBP fold: the crystal structure reveals a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dramatic change in quaternary structure.

Core-directed protein design. I. An experimental method for selecting stable proteins from combinatorial libraries.

The design of proteins represents a significant challenge to modern-day structural biology. A major obstacle here is the specification of well-packed hydrophobic cores to drive the folding and stabilization of the target. Computational approaches have been used to alleviate this by testing alternate sequences prior to the production and characterization of a few proteins. Here we present the experimental counterpart of this approach. We selected stable variants from a library of ubiquitin hydrophobic-core mutants as follows. Hexahistidine-tagged proteins were displayed on the surface of phage. These protein-phage were immobilized onto Ni-coated surfaces. The bound fusion-phage were treated with protease to remove unstable or poorly folded proteins. Stable phage fusions were eluted and infected into Escherichia coli, which allowed amplification for further selection, sequencing, or protein expression. Two Ni-derivatized supports were tested: Ni-NTA chips for surface plasmon resonance (SPR) and Ni-NTA agarose beads. SPR had an advantage in that the selection process could be monitored directly. This allowed individual clones and experimental conditions to be tested rapidly prior to preparative panning of the library, which was carried out using Ni-NTA agarose beads. We demonstrate the method by selecting stable core mutants of ubiquitin, the characterization of which is described in the following paper [Finucane, M. D., and Woolfson, D. N. (1999) Biochemistry 38, XXXXX-XXXXX]. As our method selects only on the basis of structure and stability, it will be of use in improving the stabilities and structural specificities of proteins of de novo design, and in establishing rules that link sequence and structure.

Core-directed protein design. II. Rescue of a multiply mutated and destabilized variant of ubiquitin.

We have applied the method described in the preceding paper [Finucane, M. D., et al. (1999) Biochemistry 38, 11604-11612], namely, stability-based selection using phage display, to explore the sequence requirements for packing in the hydrophobic core of ubiquitin. In contrast to the parent protein, which was a structurally compromised mutant, the selected variants could be overexpressed and purified in yields for structural studies. In particular, CD and NMR measurements showed that the selectants folded correctly to stable native-like structures. These points demonstrate the utility of our core-directed method for stabilizing and redesigning proteins. In addition and in contrast to foregoing studies on other proteins, which suggest that hydrophobic cores permit substitutions provided that hydrophobicity and core volumes are generally conserved, we find that the core of ubiquitin is surprisingly intolerant of amino acid substitutions; variants that survived our selection showed a clear consensus for the wild-type sequence. It is probable that our results differed from those from other groups for two reasons. First, ubiquitin may be unusual in that it has strict sequence requirements for its structure and stability. We discuss this result in light of sequence conservation in the eukaryotic ubiquitins and proteins of the ubiquitin structural superfamily. Second, our mutants were selected solely on the basis of stability, in contrast to the other studies that rely on function-based selection. The latter may lead to proteins that are more plastic and tolerant of substitutions.

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.

Determinants of strand register in antiparallel beta-sheets of proteins.

Antiparallel beta-sheets present two distinct environments to inter-strand residue pairs: beta(A,HB) sites have two backbone hydrogen bonds; whereas at beta(A,NHB) positions backbone hydrogen bonding is precluded. We used statistical methods to compare the frequencies of amino acid pairs at each site. Only approximately 10% of the 210 possible pairs showed occupancies that differed significantly between the two sites. Trends were clear in the preferred pairs, and these could be explained using stereochemical arguments. Cys-Cys, Aromatic-Pro, Thr-Thr, and Val-Val pairs all preferred the beta(A,NHB) site. In each case, the residues usually adopted sterically favored chi1 conformations, which facilitated intra-pair interactions: Cys-Cys pairs formed disulfide bonds; Thr-Thr pairs made hydrogen bonds; Aromatic-Pro and Val-Val pairs formed close van der Waals contacts. In contrast, to make intimate interactions at a beta(A,HB) site, one or both residues had to adopt less favored chi1 geometries. Nonetheless, pairs containing glycine and/or aromatic residues were favored at this site. Where glycine and aromatic side chains combined, the aromatic residue usually adopted the gauche conformation, which promoted novel aromatic ring-peptide interactions. This work provides rules that link protein sequence and tertiary structure, which will be useful in protein modeling, redesign, and de novo design. Our findings are discussed in light of previous analyses and experimental studies.

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

BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: The 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.

Buried polar residues and structural specificity in the GCN4 leucine zipper.

A conserved asparagine (Asn 16) buried in the interface of the GCN4 leucine zipper selectively favours the parallel, dimeric, coiled-coil structure. To test if other polar residues confer oligomerization specificity, the structural effects of Gln and Lys substitutions for Asn 16 were characterized. Like the wild-type peptide, the Asn 16Lys mutant formed exclusively dimers. In contrast, Gln 16, despite its chemical similarity to Asn, allowed the peptide to form both dimers and trimers. The Gln 16 side chain was accommodated by qualitatively different interactions in the dimer and trimer crystal structures. These findings demonstrate that the structural selectivity of polar residues results not only from the burial of polar atoms, but also depends on the complementarity of the side-chain stereochemistry with the surrounding structural environment.

A designed heterotrimeric coiled coil.

Principles that guide folding of coiled coils were tested by designing three peptides that preferentially associate with each other to form a heterotrimeric coiled coil. The core positions of the designed helices contained residues that promote formation of trimeric coiled coils. Ionic interactions were employed to mediate heterospecificity, and negative design was used to favor formation of the heterotrimer over alternative arrangements. A program was written to select sequences that maximized the number of attractive interhelical interactions in a parallel heterotrimer and the number of repulsive electrostatic interactions in alternative species. Solution studies indicate that an equimolar mixture of the three peptides forms a helical trimer with high specificity and stability. These results validate the principles used to guide the design and suggest that the heterotrimer may serve as a useful, autonomous trimerization domain.

Predicting oligomerization states of coiled coils.

An algorithm based on the profile method was developed that faithfully distinguishes between the amino acid sequences of dimeric and trimeric coiled coils. Normalized sequence profiles derived from nonhomologous, two- and three-stranded, coiled-coil sequences with unambiguous registers were used to assign dimer and trimer propensities to test sequences. The difference between the dimer and trimer profile scores accurately reflected the preferred oligomerization state. The method relied on two strategies that may be generally applicable to profile calculations--profile values of solvent-exposed residues and of amino acids that were underrepresented in the data-base were given zero weight. Differences between the dimer and trimer profiles revealed sequence patterns that match and extend experimental studies of oligomer specification.

Dissecting the structure of a partially folded protein. Circular dichroism and nuclear magnetic resonance studies of peptides from ubiquitin.

The nature and interaction of structural elements in a partially ordered form of ubiquitin, the A-state, which is populated at low pH in 40 to 60% aqueous methanol, have been investigated. Two synthetic peptides have been studied under the same conditions: U(1-21), corresponding to the N-terminal beta-hairpin in the native (N) and A-states of ubiquitin and U(1-35), which includes this hairpin plus an alpha-helix. Circular dichroism studies indicate that, although these peptides are largely unfolded in water, their structural content in 30 and 60% methanol is comparable with the corresponding native secondary structure. Sequence-specific assignments of the 1H n.m.r. spectra of U(1-35) in aqueous methanol and subsequent secondary structure determination confirm the conservation in detail of native-like secondary structure. Corresponding resonances in spectra of U(1-35), U(1-21) and the A-state itself were found to have closely similar chemical shifts, suggesting that the beta-hairpin exists independently in the partially folded protein, with little or no influence from the rest of the molecule. This is confirmed by the virtual absence in nuclear Overhauser enhancement spectroscopy and rotating frame nuclear Overhauser enhancement spectroscopy spectra of nuclear Overhauser enhancement effects between structural elements. c.d. and n.m.r. evidence suggests that structure in the C-terminal half of the molecule in the A-state is largely non-native. Thus, although methanol is necessary to assure its stability in the absence of wider native interactions, the structure of the beta-hairpin, including the register of its hydrogen bonding, appears to be determined entirely by its own sequence. This intrinsic structural preference in the first part of the ubiquitin sequence is much stronger than in the C-terminal half, a conclusion reflected in the results from a variety of secondary structure prediction algorithms.

Topological and stereochemical restrictions in beta-sandwich protein structures.

Chain topology in beta-structured protein domains and handedness associated with it are discussed. Previously, other workers have shown that by considering just two restrictions--structures that are left-handed and/or have loops that cross can be disregarded--the number of topologies associated with such structures is expected to be severely limited. By way of example, we determine the number of topologies compatible with a six-stranded antiparallel beta-sandwich. Without restriction on the type of strand-strand connection allowed but with elimination of symmetry related structures 360 topologies are possible. If connections between parallel strands are disqualified the number is reduced, 10-fold, to 36. The figure is cut to 24 when structures with loop crossings are eliminated. Handedness in these structures is examined in detail and from this a rationale for the observed predominance of right-handed forms of beta-structures is presented. The 24 structures can be considered as a set of right- and left-handed pairs of 12 topologies. All but two of these pairs can be assigned hands on the basis of existing rules. Six of the structures are found to occur in the Brookhaven Protein Databank and all are right-handed. This study provides a basis for protein design projects which might, for example, attempt the synthesis of unobserved protein topologies. Of the 24 structures in the final set eight are examples of the classic Greek key fold. Thus, the predominance of this motif among all-beta proteins can be attributed in part to these topological constraints. The possible physicochemical origins of the structural selection rules and additional factors which might contribute to the particular favourability of certain structures are also explored.