Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATPgammaS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiCWT) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.     

Hexamerization and thermostability emerged very early during geranylgeranylglyceryl phosphate synthase evolution

A large number of archaea live in hyperthermophilic environments. In consequence, their proteins need to adopt to these harsh conditions, including the enzymes that catalyze the synthesis of their membrane ether lipids. The enzyme that catalyzes the formation of the first ether bond in these lipids, geranylgeranylglyceryl phosphate synthase (GGGPS), exists as a hexamer in many hyperthermophilic archaea, and a recent study suggested that hexamerization serves for a fine‐tuning of the flexibility – stability trade‐off under hyperthermophilic conditions. We have recently reconstructed the sequences of ancestral group II GGGPS enzymes and now present a detailed biochemical characterization of nine of these predecessors, which allowed us to trace back the evolution of hexameric GGGPS and to draw conclusions about the properties of extant GGGPS branches that were not accessible to experiments up to now. Almost all ancestral GGGPS proteins formed hexamers, which demonstrates that hexamerization is even more widespread among the GGGPS family than previously assumed. Furthermore, all experimentally studied ancestral proteins showed high thermostability. Our results indicate that the hexameric oligomerization state and thermostability were present very early during the evolution of group II GGGPS, while the fine tuning of the flexibility – stability trade‐off developed very late, independent of the emergence of hexamerization.     

Clustering of OB-fold domains of the partner protease complexed with trimeric stomatin from Thermococcales

The C-terminal soluble domain of stomatin operon partner protein (STOPP) of the hyperthermophilic archaeon Pyrococcus horikoshii has an oligonucleotide binding-fold (OB-fold). STOPP lacks the conserved surface residues necessary for binding to DNA/RNA. A tryptophan (W) residue is conserved instead at the molecular surface. Solvent-accessible W residues are often found at interfaces of protein–protein complexes, which suggested the possibility of self-assembling of STOPP. Protein–protein interactions among the C-terminal soluble domains of STOPP PH1510 (1510-C) were then analyzed by chemical linking and blue native polyacrylamide gel electrophoresis (BN-PAGE) methods. These results suggest that the soluble domains of STOPP could assemble into homo-oligomers. Since hexameric subcomplex I from archaeal proteasome consists of coiled-coil segments and OB-fold domains, molecular modeling of 1510-C was performed using hexameric subcomplex I as a template. Although 1510-C is a comparatively small polypeptide consisting of approximately 60 residues, numerous salt bridges and hydrophobic interactions were observed in the predicted hexamer of 1510-C, suggesting the stability of the homo-oligomeric structure. This oligomeric property of STOPP may be favorable for triplicate proteolysis of the trimer of prokaryotic stomatin.     

A single amino acid can switch the oligomerization state of the alpha-helical coiled-coil domain of cartilage matrix protein.

We have studied the oligomerization of an alpha-helical coiled-coil using as an example a peptide corresponding to the C-terminal domain of cartilage matrix protein. By replacing one arginine residue, which forms an interchain ionic interaction with a glutamic acid residue, with glutamine, we found that this peptide assembles into a homotetramer at neutral pH in contrast to the native molecule which forms homotrimers. At acidic and basic pH, however, we again observed the trimer conformation. Another arginine, which is probably involved in an intrachain salt bridge, has no effect on the assembly. Our data demonstrate that besides the specific distribution of hydrophobic residues, interchain ionic interactions can be crucial in modulating the association behavior of alpha-helical coiled-coil domains.     

Partial proteolysis of simian virus 40 T antigen reveals intramolecular contacts between domains and conformation changes upon hexamer assembly

Simian virus 40 large tumor antigen (Tag) is a multi-functional viral protein that binds specifically to SV40 origin DNA, serves as the replicative DNA helicase, and orchestrates the assembly and operation of the viral replisome. Tag associated with Mg-ATP forms hexamers and, in the presence of SV40 origin DNA, double hexamers. Limited tryptic digestion of monomeric Tag revealed three major stable structural domains. The N-terminal domain spans amino acids 1-130, the central domain comprises amino acids 131-476, and the C-terminal domain extends from amino acid 513 to amino acid 698. Co-immunoprecipitation of digestion products of monomeric Tag suggests that the N-terminal domain associates stably with sequences located in the central region of the same Tag molecule. Hexamer formation protected the tryptic cleavage sites in the exposed region between the central and C-terminal domains. Upon hexamerization, this exposed region also became less accessible to a monoclonal antibody whose epitope maps in that region. The tryptic digestion products of the soluble hexamer and the DNA-bound double hexamer were indistinguishable. A low-resolution model of the intramolecular and intermolecular interactions among Tag domains in the double hexamer is proposed.     

Association Equilibrium of the HIV-1 Capsid Protein in a Crowded Medium Reveals that Hexamerization during Capsid Assembly Requires a Functional C-Domain Dimerization Interface

Polymerization of the intact capsid protein (CA) of HIV-1 into mature capsidlike particles at physiological ionic strength in vitro requires macromolecularly crowded conditions that approach those inside the virion, where the mature capsid is assembled in vivo. The capsid is organized as a hexameric lattice. CA subunits in each hexamer are connected through interfaces that involve the CA N-terminal domain (NTD); pairs of CA subunits belonging to different hexamers are connected through a different interface that involves the C-terminal domain (CTD). At physiological ionic strength in noncrowded conditions, CA subunits homodimerize through this CTD-CTD interface, but do not hexamerize through the other interfaces (those involving the NTD). Here we have investigated whether macromolecular crowding conditions are able to promote hexamerization of the isolated NTD and/or full-length CA (with an inactive CTD-CTD interface to prevent polymerization). The oligomerization state of the proteins was determined using analytical ultracentrifugation in the absence or presence of high concentrations of an inert macromolecular crowding agent. Under the same conditions that promoted efficient assembly of intact CA dimers, neither NTD nor CA with an inactive CTD-CTD interface showed any tendency to form hexamers or any other oligomer. This inability to hexamerize was observed even in macromolecularly crowded conditions. The results indicate that a functional CTD-CTD interface is strictly required for hexamerization of HIV-1 CA through the other interfaces. Together with previous results, these observations suggest that establishment of NTD-CTD interactions involved in CA hexamerization during mature HIV-1 capsid assembly requires a homodimerization-dependent conformational switching of CTD.     

Oligomerization of the chromatin-structuring protein H-NS

H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.     

Mapping of structural determinants for the oligomerization of p58, a lectin-like protein of the intermediate compartment and cis-Golgi.

Shortly after synthesis, p58, the rat homologue of the mannose-binding lectin ERGIC-53/MR60, which localizes to pre-Golgi and cis-Golgi compartments, forms dimers and hexamers, after which an equilibrium of both forms is reached. Mature p58, a type I membrane protein, contains four cysteine residues in the lumenal domain which are capable of forming disulphide bonds. The membrane-proximal half of the lumenal domain consists of four predicted alpha-helical domains, one heavily charged and three amphipathic in nature, all candidates for electrostatic or coiled-coil interactions. Using single-stranded mutagenesis, the cysteines were individually changed to alanines and the contribution of each of the alpha-helical domains was probed by internal deletions. The N-terminal cysteine to alanine mutants, C198A and C238A and the double mutant, C198/238A, oligomerized like the wild-type protein. The two membrane-proximal cysteines were found to be necessary for the oligomerization of p58. Mutants lacking one of the membrane proximal cysteines, either C473A or C482A, were unable to form hexamers, while dimers were formed normally. The C473/482A double mutant formed only monomers. Deletion of any of the individual alpha-helical domains had no effect on oligomerization. The dimeric and hexameric forms bound equally well to D-mannose. The dimeric and monomeric mutants displayed a cellular distribution similar to the wild-type protein, indicating that the oligomerization status played a minimal role in maintaining the subcellular distribution of p58.     

YspD: A Potential Therapeutic Target for Drug Design to Combat Yersinia enterocolitica Infection

YspD is an annotated hydrophilic translocator of Ysa–Ysp type III secretion system of Yersinia enterocolitica. YspD has sequence, secondary structure and three-dimensional structure similar to other hydrophilic translocators. All hydrophilic translocators lack transmembrane region and possess intramolecular coiled-coil region. Disordered regions are mostly clustered at the N-terminal. Large loops provide flexibility, allowing conformational changes during oligomerization and protein–protein interaction. LcrV and PcrV have globular N-terminal and C-terminal domains, connected by intramolecular coiled-coil region. YspD, IpaD, SipD and BipD lack globular N-terminal and C-terminal domains. Their N-terminal and C-terminal domain have a bundle like structure connected by the intramolecular coiled-coil. The intramolecular coiled-coil regions (helix-5&9) of YspD showed maximum conservation, followed by helices at N-terminal. Polar interactions are mainly involved during dimerization of YspD, involving polar residues from helix-9 of both the YspD molecules. A methionine forms the boundary of interaction between the two YspD molecules. The two YspD molecules are arranged in antiparallel fashion to form the dimer. N-terminal of YspB interacted with C-terminal of YspD molecule to form a pentameric complex, consisting four YspD molecules and one YspB molecule. Sequence, structural similarity and presence of specific motifs in YspD (like chaperone protein) indicate the ability of N-terminal domain to show self-chaperoning activity and regulate folding and conformational state of YspD during its journey from the bacterial cytoplasm to the needle tip. Structural analysis of YspD and its mechanism of interaction with other proteins would enable us to design drugs against this hydrophilic protein to combat Yersinia infection.

     

Solution structure of the C-terminal antiparallel coiled-coil domain from Escherichia coli osmosensor ProP

Bacteria respond to increasing medium osmolality by accumulating organic solutes that are compatible with cellular functions. Transporter ProP of Escherichia coli, a proton symporter and a member of the major facilitator superfamily, senses osmotic shifts and responds by importing osmolytes such as glycine betaine. ProP contains a cytoplasmic, C-terminal extension that is essential for its activity. A peptide corresponding to the C-terminal extension of ProP forms a homodimeric alpha-helical coiled-coil even though some of its heptad a positions are not occupied by hydrophobic amino acid residues. Unexpectedly, amino acid replacement R488I, occurring at a heptad a position, destabilized the coiled-coil formed by the ProP peptide and attenuated the response of the intact transporter to osmotic upshifts in vivo. Thus, ProP was proposed to dimerize via an antiparallel coiled-coil. We used nuclear magnetic resonance (NMR) spectroscopy to determine the structure of the synthetic peptide corresponding to residues 468-497 of ProP. This region did form an antiparallel coil-coil in which critical residue R488 specifies the antiparallel coiled-coil orientation by forming stabilizing salt-bridges. Charged residues (both acidic and basic) are clustered on the c/g surface of the coiled-coil whereas polar residues are distributed on the b/e surface. This causes the structure to be bent, in contrast to other known antiparallel coiled-coils (those from the hepatitis delta antigen (PDB ID code 1A92) and the bovine F(1) ATPase inhibitor protein (PDB ID code 1HF9)). The coiled-coil and its possible importance for osmosensing are discussed.     

Structural properties and evolutionary relationships of PspA, a surface protein of Streptococcus pneumoniae, as revealed by sequence analysis.

Analysis of the sequence for the gene encoding PspA (pneumococcal surface protein A) of Streptococcus pneumoniae revealed the presence of four distinct domains in the mature protein. The structure of the N-terminal half of PspA was highly consistent with that of an alpha-helical coiled-coil protein. The alpha-helical domain was followed by a proline-rich domain (with two regions in which 18 of 43 and 5 of 11 of the residues are prolines) and a repeat domain consisting of 10 highly conserved 20-amino-acid repeats. A fourth domain consisting of a hydrophobic region too short to serve as a membrane anchor and a poorly charged region followed the repeats and preceded the translation stop codon. The C-terminal region of PspA did not possess features conserved among numerous other surface proteins, suggesting that PspA is attached to the cell by a mechanism unique among known surface proteins of gram-positive bacteria. The repeat domain of PspA was found to have significant homology with C-terminal repeat regions of proteins from Streptococcus mutans, Streptococcus downei, Clostridium difficile, and S. pneumoniae. Comparisons of these regions with respect to functions and homologies suggested that, through evolution, the repeat regions may have lost or gained a mechanism for attachment to the bacterial cell.     

The hexameric structures of human heat shock protein 90

Background

The human 90-kDa heat shock protein (HSP90) functions as a dimeric molecular chaperone. HSP90 identified on the cell surface has been found to play a crucial role in cancer invasion and metastasis, and has become a validated anti-cancer target for drug development. It has been shown to self-assemble into oligomers upon heat shock or divalent cations treatment, but the functional role of the oligomeric states in the chaperone cycle is not fully understood.

Principal Findings

Here we report the crystal structure of a truncated HSP90 that contains the middle segment and the carboxy-terminal domain, termed MC-HSP90. The structure reveals an architecture with triangular bipyramid geometry, in which the building block of the hexameric assembly is a dimer. In solution, MC-HSP90 exists in three major oligomer states, namely dimer, tetramer and hexamer, which were elucidated by size exclusion chromatography and analytical ultracentrifugation. The newly discovered HSP90 isoform HSP90N that lacks the N-terminal ATPase domain also exhibited similar oligomerization states as did MC-HSP90.

Conclusions

While lacking the ATPase domain, both MC-HSP90 and HSP90N can self-assemble into a hexameric structure, spontaneously. The crystal structure of MC-HSP90 reveals that, in addition to the C-terminal dimerization domain, the residue W320 in the M domain plays a critical role in its oligomerization. This study not only demonstrates how the human MC-HSP90 forms a hexamer, but also justifies the similar formation of HSP90N by using 3D modeling analysis.     

Novel hexamerization motif is discovered in a conserved cytoplasmic protein from <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

The cytoplasmic protein Stm3548 of unknown function obtained from a strain of Salmonella typhimurium was determined by X-ray crystallography at a resolution of 2.25 A. The asymmetric unit contains a hexamer of structurally identical monomers. The monomer is a globular domain with a long beta-hairpin protrusion that distinguishes this structure. This beta-hairpin occupies a central position in the hexamer, and its residues participate in the majority of interactions between subunits of the hexamer. We suggest that the structure of Stm3548 presents a new hexamerization motif. Because the residues participating in interdomain interactions are highly conserved among close members of protein family DUF1355 and buried solvent accessible area for the hexamer is significant, the hexamer is most likely conserved as well. A light scattering experiment confirmed the presence of hexamer in solution.     

Coiled coils: a highly versatile protein folding motif

The alpha-helical coiled coil is one of the principal subunit oligomerization motifs in proteins. Its most characteristic feature is a heptad repeat pattern of primarily apolar residues that constitute the oligomer interface. Despite its simplicity, it is a highly versatile folding motif: coiled-coil-containing proteins exhibit a broad range of different functions related to the specific 'design' of their coiled-coil domains. The architecture of a particular coiled-coil domain determines its oligomerization state, rigidity and ability to function as a molecular recognition system. Much progress has been made towards understanding the factors that determine coiled-coil formation and stability. Here we discuss this highly versatile protein folding and oligomerization motif with regard to its structural architecture and how this is related to its biological functions.     

The role of the carboxyl terminal alpha-helical coiled-coil domain in osmosensing by transporter ProP of Escherichia coli

Concentrative uptake of osmoprotectants via transporter ProP contributes to the rehydration of Escherichia coli cells that encounter high osmolality media. A member of the major facilitator superfamily, ProP is activated by osmotic upshifts in whole bacteria, in cytoplasmic membrane vesicles and in proteoliposomes prepared with the purified protein. Soluble protein ProQ is also required for full osmotic activation of ProP in vivo. ProP is differentiated from structural and functional homologues by its osmotic activation and its C-terminal extension, which is predicted to form an alpha-helical coiled-coil. A synthetic polypeptide corresponding to the C-terminus of ProP (ProP-p) formed a dimeric alpha-helical coiled-coil. A derivative of transporter ProP lacking 26 C-terminal amino acids was expressed but inactive. A derivative harbouring amino acid changes K460I, Y467I and H495I (each at the core, coiled-coil 'a' position) required a larger osmotic upshift for activation than did the wild type transporter. The same changes extended, stabilized and altered the oligomeric state of the coiled-coil formed by ProP-p. Amino acid change R488I (also at the 'a' position) further increased the magnitude of the osmotic upshift required to activate ProP, reduced the activity attained and rendered ProP activation transient. Unexpectedly, replacement R488I destabilized the coiled-coil formed by ProP-p. The activity and osmotic activation of ProP were even more strongly attenuated by helix-destabilizing change I474P. These data demonstrate that the carboxyl terminal domain of ProP can form a homodimeric alpha-helical coiled-coil with unusual properties. They implicate the C-terminal domain in the osmotic activation of ProP.     

The linker region between the helicase and primase domains of the gene 4 protein of bacteriophage T7. Role in helicase conformation and activity

The gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for template-directed oligoribonucleotide synthesis. A 26 amino acid linker region (residues 246-271) connects the two domains and is essential for the formation of functional hexamers. In order to further dissect the role of the linker region, three residues (Ala257, Pro259, and Asp263) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 phage lacking gene 4. The in vivo screening revealed Ala257 and Asp263 to be essential whereas Pro259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala257 or Asp263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg2+, all of the altered proteins oligomerize. However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation. Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA. Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required. The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer.     

Insights into the oligomeric states, conformational changes, and helicase activities of SV40 large tumor antigen

The large T (LT) antigen encoded by SV40 virus is a multi-domain, multi-functional protein that can not only transform cells but can also function as an efficient molecular machine to unwind duplex DNA for DNA replication. Here we report our findings on the oligomeric forms, domain interactions, and ATPase and helicase activities of various LT constructs. For the LT constructs that hexamerize, only two oligomeric forms, hexameric and monomeric, were detected in the absence of ATP/ADP. However, the presence of ATP/ADP stabilizes LT in the hexameric form. The LT constructs lacking the N- and C-terminal domains, but still retaining hexamerization ability, have ATPase as well as helicase activities at a level comparable to the full-length LT, suggesting the importance of hexamerization for these activities. The domain structures and the possible interactions between different LT fragments were probed with limited protease (trypsin) digestion. Such protease digestion generated a distinct pattern in the presence and absence of ATP/ADP and Mg(2+). The most C-terminal fragment (residues 628-708, containing the host-range domain), which was thought to be completely unstructured, was somewhat trypsin-resistant despite the presence of multiple Arg and Lys, possibly due to a rather structured C terminus. Furthermore, the N- and C-terminal fragments cleaved by trypsin were associated with other parts of the molecule, suggesting the interdomain interactions for the fragments at both ends.     

MCM ring hexamerization is a prerequisite for DNA-binding

The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings by showing that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in the hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.     

Sequence comparisons of intermediate filament chains: evidence of a unique functional/structural role for coiled-coil segment 1A and linker L1

A comprehensive analysis of the sequences of all types of intermediate filament chains has been undertaken with a particular emphasis on those of segment 1A and linker L1. This has been done to assess whether structural characteristics can be recognized in the sequences that would be consistent with the role of each region in the recently proposed "swinging head" hypothesis. The analyses show that linker L1 is the most flexible rod domain region, that it is the most elongated structure (on a per residue basis), and that it is the most variable region as regards sequence and length. Segment 1A has one of the two most highly conserved regions of sequence in the rod domain (the other being at the end of segment 2B), with seven particular residues conserved across all chain types. It also contains one of the very few potential interchain ionic interactions that could be conserved across all chain types. However, the aggregation of chains in segment 1A is specified less precisely overall by interchain ionic interactions than are the other coiled-coil segments. The apolar residue contents in positions a and d of the heptad substructure are the highest of any coiled-coil segment in the intermediate filament family. Segment 1A also displays an amino acid composition atypical of not only coiled-coil segments 1B and 2B, but indeed of two-stranded coiled coils in general. Nonetheless, molecular modeling based on the crystal structure of the monomeric 1A fragment from human vimentin shows that coiled-coil formation is plausible. The most extensive regions of apolar/aromatic residues lie at the C-terminal end of segment 2B in the helix termination motif and in segment 1A in and close to the helix initiation motif. The predicted stability of the individual alpha-helices in segment 1A is greater than in those comprising segments 1B and 2B, though potential intrachain ionic interactions are either lacking or are minimal in number. Analysis of the 1A sequence and those regions immediately N- and C-terminal to it has shown that the capping residues are near optimal close to the previously predicted ends, thus adding to the likely stability of the alpha-helical structure. However, a second terminating sequence is predicted in 1A (about 10 residues back from the C-terminus). This allows the possibility of some unwinding of the alpha-helical structure of 1A immediately adjacent to linker L1 when the head domains no longer stabilize the coiled-coil structure. All of these data are consistent with the concept of a flexible hinge at L1 and with the ability of the two alpha-helical coiled-coil strands to separate under appropriate conditions and partly unwind at their C-terminal ends to allow the head domains a greater degree of mobility, thus facilitating function.