Oligomerization-dependent changes in the thermodynamic properties of the TPR-MET receptor tyrosine kinase

Although oligomerization of receptor tyrosine kinases (RTKs) is necessary for receptor activation and signaling, a quantitative understanding of how oligomerization mediates these critical processes does not exist. We present a comparative thermodynamic analysis of functionally active dimeric and functionally inactive monomeric soluble analogues of the c-MET RTK, which clearly reveal that oligomerization regulates the binding affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide substrates. Thermodynamic binding data for oligomeric c-MET were obtained from the dimeric TPR-MET oncoprotein, a functionally active fusion derivative of the c-MET RTK. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil dimerization domain from the nuclear pore complex. Comparative data were obtained from a soluble monomeric kinase compromising the c-MET cytoplasmic domain (cytoMET). Significantly, under equilibrium binding conditions, the oligomeric phosphorylated kinase showed a significantly lower dissociation constant (K(d,dimer) = 11 microM) for a tyrosine-containing peptide derived from the C-terminal tail of the c-MET RTK when compared to the phosphorylated monomeric kinase cytoMET (K(d,monomer) = 140 microM). Surprisingly, equilibrium dissociation constants measured for the kinase and ATP were independent of the oligomerization state of the kinase (approximately 10 microM). Stopped-flow analysis of peptide substrate binding showed that the association rate constants (k(2)) differed 2-fold and dissociation rate constants (k(-2)) differed 10-fold when phosphorylated TPR-MET was compared to phosphorylated cytoMET. ATP binding abrogated the differences in k(2) rates observed between the two oligomeric states of the c-MET cytoplasmic domain. These results clearly imply that oligomerization induces important thermodynamic and conformational changes in the substrate binding regions of the c-MET protein and provide quantitative mechanistic insights into the necessary role of oligomerization in RTK activation.     

Noncovalent interactions of the Apple 4 domain that mediate coagulation factor XI homodimerization

The Apple 4 (A4) domain of human plasma factor XI (FXI) was used to investigate the process of FXI noncovalent dimer formation. Recombinant 6-histidine-tagged A4 domain proteins were prepared utilizing a bacterial expression system. Purification was accomplished under denaturing conditions, followed by a refolding protocol to facilitate correct disulfide bond formation. Analysis of the A4 domain (C321S mutant) by size exclusion chromatography indicated the presence of a slowly equilibrating reversible monomer-dimer equilibrium. The elution profiles reveal highly symmetrical peaks for both dimeric and monomeric species with elution times that were highly reproducible for varying amounts of both the dimeric and monomeric species. The monomer-dimer equilibrium was found to be dependent upon changes in both pH and salt concentration. Under conditions approximating physiologic salt concentration and pH (20 mm HEPES, 100 mm NaCl, and 1 mm EDTA, pH 7.4), it was determined that the monomer-dimer equilibrium was characterized by a dissociation constant (K(D)) value of 229 +/- 26 nm with a calculated Delta G value of 9.1 kcal/mol. This report identifies electrostatic contributions and the presence of a hydrophobic component that mediate interactions at the A4 domain interface. The rate of dissociation for the recombinant A4 domain C321S mutant was examined by monitoring the increase in 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt fluorescence under dissociating conditions, giving a value for a dissociation rate constant (k(off)) of 4.3 x 10(-3) s(-1).     

Monomeric state of S100P protein: Experimental and molecular dynamics study

S100 proteins constitute a large subfamily of the EF-hand superfamily of calcium binding proteins. They possess one classical EF-hand Ca²⁺-binding domain and an atypical EF-hand domain. Most of the S100 proteins form stable symmetric homodimers. An analysis of literature data on S100 proteins showed that their physiological concentrations could be much lower than dissociation constants of their dimeric forms. It means that just monomeric forms of these proteins are important for their functioning. In the present work, thermal denaturation of apo-S100P protein monitored by intrinsic tyrosine fluorescence has been studied at various protein concentrations within the region from 0.04–10 μM. A transition from the dimeric to monomeric form results in a decrease in protein thermal stability shifting the mid-transition temperature from 85 to 75 °C. Monomeric S100P immobilized on the surface of a sensor chip of a surface plasmon resonance instrument forms calcium dependent 1 to 1 complexes with human interleukin-11 (equilibrium dissociation constant 1.2 nM). In contrast, immobilized interleukin-11 binds two molecules of dimeric S100P with dissociation constants of 32 nM and 288 nM. Since effective dissociation constant of dimeric S100P protein is very low (0.5 μM as evaluated from our data) the sensitivity of the existing physical methods does not allow carrying out a detailed study of S100P monomer properties. For this reason, we have used molecular dynamics methods to evaluate structural changes in S100P upon its transition from the dimeric to monomeric state. 80-ns molecular dynamics simulations of kinetics of formation of S100P, S100B and S100A11 monomers from the corresponding dimers have been carried out. It was found that during the transition from the homo-dimer to monomer form, the three S100 monomer structures undergo the following changes: (1) the helices in the four-helix bundles within each monomer rotate in order to shield the exposed non-polar residues; (2) almost all lost contacts at the dimer interface are substituted with equivalent and newly formed interactions inside each monomer, and new stabilizing interactions are formed; and (3) all monomers recreate functional hydrophobic cores. The results of the present study show that both dimeric and monomeric forms of S100 proteins can be functional.     

NMR solution structure of a highly stable de novo heterodimeric coiled-coil

The NMR solution structure of a highly stable coiled-coil IAAL-E3/K3 has been solved. The E3/K3 coiled-coil is a 42-residue de novo designed coiled-coil comprising three heptad repeats per subunit, stabilized by hydrophobic contacts within the core and electrostatic interactions at the interface crossing the hydrophobic core which direct heterodimer formation. This E3/K3 domain has previously been shown to have high alpha-helical content as well as possessing a low dissociation constant (70 nM). The E3/K3 structure is completely alpha-helical and is an archetypical coiled-coil in solution, as determined using a combination of (1)H-NOE and homology based structural restraints. This structure provides a structural framework for visualizing the important interactions for stability and specificity, which are key to protein engineering applications such as affinity purification and de novo design.     

Role of intramolecular disulfides in stability and structure of a noncovalent homodimer

The importance of intramolecular disulfides in a noncovalent dimeric protein interleukin-8 (IL-8) has been studied by replacing cysteines in each of the two disulfide pairs with alpha-aminobutyric acid (CH(2)-SH --> CH(2)-CH(3)). Both disulfide mutants are less stable and exist as molten globules in the monomeric state. Interestingly, both mutants dimerize, though with slightly lower affinities compared to the native protein. NMR studies suggest a molten globule-like structure also in the dimeric state. Structures, sequence analysis, and mutagenesis studies have shown that the conserved hydrophobic residues are packed against each other in the protein core and that H bonding and van der Waals interactions stabilize the dimer interface. Deleting either disulfide in IL-8 results in substantial loss in receptor activity, indicating that both disulfides are critical for function in the folded protein. These data together suggest that the packing interactions of the hydrophobic core determine IL-8 monomer fold, that disulfides play only a marginal role in dimer formation, and that the stability imparted by the disulfides is intimately coupled to fold and function.     

Role of N‐glycosylation in EGFR ectodomain ligand binding

The epidermal growth factor receptor (EGFR) is a tyrosine kinase protein, overexpressed in several cancers. The extracellular domain of EGFR is known to be heavily glycosylated. Growth factor (mostly epidermal growth factor or EGF) binding activates EGFR. This occurs by inducing the transition from the autoinhibited tethered conformation to an extended conformation of the monomeric form of EGFR and by stabilizing the flexible preformed dimer. Activated EGFR adopts a back‐to‐back dimeric conformation after binding of another homologous receptor to its extracellular domain as the dimeric partner. Several antibodies inhibit EGFR by targeting the growth factor binding site or the dimeric interfaces. Glycosylation has been shown to be important for modulating the stability and function of EGFR. Here, atomistic MD simulations show that N‐glycosylation of the EGFR extracellular domain plays critical roles in the binding of growth factors, monoclonal antibodies, and the dimeric partners to the monomeric EGFR extracellular domain. N‐glycosylation results in the formation of several noncovalent interactions between the glycans and EGFR extracellular domain near the EGF binding site. This stabilizes the growth factor binding site, resulting in stronger interactions (electrostatic) between the growth factor and EGFR. N‐glycosylation also helps maintain the dimeric interface and plays distinct roles in binding of antibodies to spatially separated epitopes of the EGFR extracellular domain. Analysis of SNP data suggests the possibility of altered glycosylation with functional consequences. Proteins 2017; 85:1529–1549. © 2017 Wiley Periodicals, Inc.     

Leukocyte function-associated antigen 1-mediated adhesion stability is dynamically regulated through affinity and valency during bond formation with intercellular adhesion molecule-1

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the beta(2) integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.     

A structural mechanism of integrin alpha(IIb)beta(3) "inside-out" activation as regulated by its cytoplasmic face

Activation of the ligand binding function of integrin heterodimers requires transmission of an "inside-out" signal from their small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alpha(IIb)beta(3) has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the beta cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin that are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the beta cytoplasmic tails restrains the integrin in a resting state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation.     

In Silico Analysis Reveals Sequential Interactions and Protein Conformational Changes during the Binding of Chemokine CXCL-8 to Its Receptor CXCR1

Chemokine CXCL-8 plays a central role in human immune response by binding to and activate its cognate receptor CXCR1, a member of the G-protein coupled receptor (GPCR) family. The full-length structure of CXCR1 is modeled by combining the structures of previous NMR experiments with those from homology modeling. Molecular docking is performed to search favorable binding sites of monomeric and dimeric CXCL-8 with CXCR1 and a mutated form of it. The receptor-ligand complex is embedded into a lipid bilayer and used in multi ns molecular dynamics (MD) simulations. A multi-steps binding mode is proposed: (i) the N-loop of CXCL-8 initially binds to the N-terminal domain of receptor CXCR1 driven predominantly by electrostatic interactions; (ii) hydrophobic interactions allow the N-terminal Glu-Leu-Arg (ELR) motif of CXCL-8 to move closer to the extracellular loops of CXCR1; (iii) electrostatic interactions finally dominate the interaction between the N-terminal ELR motif of CXCL-8 and the EC-loops of CXCR1. Mutation of CXCR1 abrogates this mode of binding. The detailed binding process may help to facilitate the discovery of agonists and antagonists for rational drug design.     

Dissociation of the dimeric SecA ATPase during protein translocation across the bacterial membrane

The ATPase SecA mediates post-translational translocation of precursor proteins through the SecYEG channel of the bacterial inner membrane. We show that SecA, up to now considered to be a stable dimer, is actually in equilibrium with a small fraction of monomers. In the presence of membranes containing acidic phospholipids or in certain detergents, SecA completely dissociates into monomers. A synthetic signal peptide also affects dissociation into monomers. In addition, conversion into the monomeric state can be achieved by mutating a small number of residues in a dimeric and fully functional SecA fragment. This monomeric SecA fragment still maintains strong binding to SecYEG in the membrane as well as significant in vitro translocation activity. Together, the data suggest that the SecA dimer dissociates during protein translocation. Since SecA contains all characteristic motifs of a certain class of monomeric helicases, and since mutations in residues shared with the helicases abolish its translocation activity, SecA may function in a similar manner.     

Moving a microtubule may require two heads: a kinetic investigation of monomeric Ncd

Ncd is a minus-end-directed microtubule motor and a member of the kinesin superfamily. The Ncd dimer contains two motor domains, and cooperative interactions between the heads influence the interactions of each respective motor domain with the microtubule. The approach we have taken to understand the cooperativity between the two motor domains is to analyze the ATPase cycle of dimeric MC1 and monomeric MC6. The steps in the ATPase cycle where cooperativity occurs can be identified by comparing the two mechanisms. The rate-limiting step in the MC6 mechanism is ADP release at 3.4 s(-)(1). The observed rate constant for ATP-induced dissociation from the microtubule is 14 s(-)(1). However, the relative amplitude associated with MC6 dissociation is extremely small in comparison to the amplitude associated with dimeric MC1 dissociation kinetics. The amplitude data indicate that monomeric MC6 does not detach from the microtubule during the initial turnovers of ATP, and ATP hydrolysis is uncoupled from movement. The results show that cooperative interactions between the motor domains of the dimer are required for ATP-dependent dissociation; therefore, one function of the partner motor domain may be to weaken the interaction of the adjacent head with the microtubule.     

Physico-chemical properties of molten dimer ascorbate oxidase

The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 x 10(8) Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DeltaV approximately -200 mL.mol(-1)) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (DeltaG approximately 2 kcal.mol(-1)) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.     

Effect of protein kinase A-mediated phosphorylation on the structure and association properties of the enteropathogenic Escherichia coli Tir virulence protein

Enteropathogenic Escherichia coli virulence is dependent on delivery of the translocated intimin receptor protein (Tir) into host cells. Tir phosphorylation on a single tyrosine (Tyr-474) is essential in mediating cytoskeletal rearrangement correlated with disease. Tir is also phosphorylated on other residues, with cAMP-dependent kinase (PKA) modification shown to play a role in Tir function. However, the mechanism by which nontyrosine phosphorylation affects Tir function remains unclear. In this study, analytical ultracentrifugation, SDS and native gel electrophoresis revealed that both Tir and its C-terminal domain (residues 385-550 of Tir that include the PKA substrate sites) exist in an equilibrium of monomers, dimers, and in the case of Tir, higher oligomers. PKA phosphorylation (1:300, PKA/C-Tir, mol/mol) shifted the equilibrium of C-Tir, but not Tir, predominantly to the dimeric state, whereas, at 100-fold higher concentrations of PKA the phosphorylated C-Tir was largely monomeric. This monomeric state was also produced at the lower PKA concentration and physiological ionic strength. Phosphorylation-mediated effects were achieved without significant changes in secondary structure as determined by circular dichroism spectroscopy. The data presented indicate that PKA-mediated phosphorylation induces changes in the association properties of the C-terminal domain of Tir that may facilitate Tir-Tir interactions and subsequently C-Tir-host protein interactions in vivo.     

Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis

The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Ų. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcal protein A, designated domain B, was chemically synthesized. This gene was tandemly repeated to give dimeric and tetrameric domain B genes by the use of two restriction enzymes which gave blunt ends. The genes were highly expressed in Escherichia coli to afford a large amount of dimeric and tetrameric domain B proteins. The single domain B protein was efficiently produced as a fusion protein with a salmon growth hormone fragment. The fusion protein was converted to monomeric domain B by cyanogen bromide cleavage. The CD spectra of the monomeric, dimeric and tetrameric domain B proteins were essentially the same as that of native form protein A, showing that their secondary structures were very similar. The dimeric and tetrameric domain B proteins formed precipitates with IgG as protein A. This system permits the efficient production of mutated single and multiple IgG-binding domains which can be used to study structural changes and protein A-immunoglobulin interactions.     

Regulation of kinase and intermolecular bonding in intact and truncated epidermal growth factor receptor

Tyrosine kinase activity of the epidermal growth factor (EGF) receptor can be regulated by its state of association. Studies done with the purified receptor solubilized in Triton X-100 indicate that dimer formation results in negative regulation of kinase, whereas successive binding of EGF and ATP shift the association equilibrium toward the catalytically active monomeric form. The promotion of the monomeric state by ATP can be mimicked by various nonphosphorylating analogs indicating that nucleotide binding rather than autophosphorylation is responsible for stabilizing the monomeric receptor form. Truncated receptor forms, lacking either the external EGF-binding domain or the internal kinase (ATP-binding) domain, are unable to form stable dimers. These results suggest that both intra- and extracellular domains of the receptor act to stabilize the kinase-regulatory dimer.     

Dimer dissociation and unfolding mechanism of coagulation factor XI apple 4 domain: spectroscopic and mutational analysis

The blood coagulation protein factor XI (FXI) consists of a pair of disulfide-linked chains each containing four apple domains and a catalytic domain. The apple 4 domain (A4; F272-E362) mediates non-covalent homodimer formation even when the cysteine involved in an intersubunit disulfide is mutated to serine (C321S). To understand the role of non-covalent interactions stabilizing the FXI dimer, equilibrium unfolding of wild-type A4 and its C321S variant was monitored by circular dichroism, intrinsic tyrosine fluorescence and dynamic light scattering measurements as a function of guanidine hydrochloride concentration. Global analysis of the unimolecular unfolding transition of wild-type A4 revealed a partially unfolded equilibrium intermediate at low to moderate denaturant concentrations. The optically detected equilibrium of C321S A4 also fits best to a three-state model in which the native dimer unfolds via a monomeric intermediate state. Dimer dissociation is characterized by a dissociation constant, K(d), of approximately 90 nM (in terms of monomer), which is in agreement with the dissociation constant measured independently using fluorescence anisotropy. The results imply that FXI folding occurs via a monomeric equilibrium intermediate. This observation sheds light on the effect of certain naturally occurring mutations, such as F283L, which lead to intracellular accumulation of non-native forms of FXI. To investigate the structural and energetic consequences of the F283L mutation, which perturbs a cluster of aromatic side-chains within the core of the A4 monomer, it was introduced into the dissociable dimer, C321S A4. NMR chemical shift analysis confirmed that the mutant can assume a native-like dimeric structure. However, equilibrium unfolding measurements show that the mutation causes a fourfold increase in the K(d) value for dissociation of the native dimer and a 1 kcal/mol stabilization of the monomer, resulting in a highly populated intermediate. Since the F283 side-chain does not directly participate in the dimer interface, we propose that the F283L mutation leads to increased dimer dissociation by stabilizing a monomeric state with altered side-chain packing that is unfavorable for homodimer formation.     

The Small Myelin-Associated Glycoprotein Is a Zinc-Binding Protein

The myelin-associated glycoprotein is a transmembrane cell adhesion molecule expressed specifically by myelinating glial cells of the nervous system. Its two isoforms, whose amino acid sequences differ only by their respective cytoplasmic carboxy-terminal domains, are important for the formation and maintenance of a normal functional myelin sheath. In this study, by using recombinant proteins, we identify the cytoplasmic domain of the small isoform of the myelin-associated glycoprotein as a zinc-binding protein. The observed dissociation constant lies in the low micromolar range (K(D) = 6-7 microM). The binding of zinc by the small myelin-associated glycoprotein induces a conformational change that enables the protein to reversibly bind to a hydrophobic phenyl-Sepharose matrix. Our results also suggest that zinc may induce dimerization of the small myelin-associated glycoprotein. We suggest roles for zinc in the stabilization of the structure of the cytoplasmic domain of the small myelin-associated glycoprotein and in protein-protein interactions that involve this short domain.     

Molecular dynamics simulations of human cystatin C and its L68Q varient to investigate the domain swapping mechanism

Human cystatin C variant (L68Q), one of the amyloidgenic proteins, has been shown to form dimeric structure spontaneously via domain swapping and easily cause amyloid deposits in the brains of patients suffering from Alzheimer's disease or hereditary cystatin C amyloid angiopathy. The monomeric L68Q and wild-type (wt) HCCs share similar structural feature consisting of a core with a five-stranded anti-parallel beta-sheet (beta-region) wrapped around a central helix. In this study, various molecular dynamics simulations were conducted to investigate the conformational fluctuations of the monomeric L68Q and wt HCCs at various combinations of temperature (300 and 500K) and pH (2 and 7) to gain insights into the domain swapping mechanism. The results show that elevated temperature accelerates the disruption of the hydrophobic core and acidic condition promotes the destruction of three salt bridges between beta2 and beta3 in both HCCs. The results also indicate that the interior hydrophobic core of the L68Q variant is relatively unstable, leading to domain swapping more readily comparing to wt HCC under conditions favoring this process. However, these two monomeric HCCs adopt the same mechanism of domain swapping as follows: (i) first, the interior hydrophobic core is disrupted; (ii) subsequently, the central helix departs from the beta-region; (iii) then, the beta2-L1-beta3 hairpin structure unfolds following the so-called "zip-up" mechanism; and (iv) finally, the open form HCC is generated.