Structural characterization of the cytosolic domain of kidney chloride/bicarbonate anion exchanger 1 (kAE1)

Kidney anion exchanger 1 (kAE1) is a membrane glycoprotein expressed in alpha-intercalated cells in the collecting ducts of the kidney where it mediates electroneutral chloride/bicarbonate exchange. Human kAE1 is a truncated form of erythroid AE1 missing the first 65 residues of the N-terminal cytosolic domain, which includes a disordered acidic region (residues 1-54) and the first beta-strand (residues 55-65) of the folded region. Unlike erythroid AE1, kAE1 does not bind deoxyhemoglobin, glycolytic enzymes, or cytoskeletal components. To understand the effect of the N-terminal deletion on the structure of the cytosolic domain, we performed an extensive biophysical analysis on His 6 tagged cytosolic domains of erythroid AE1 (cdAE1), kidney AE1 (cdkAE1), and a novel truncation mutant (cdDelta54AE1) missing the first 54 residues, but retaining the beta-strand. Circular dichroism did not detect any major differences in secondary structure, and sedimentation analyses showed that all three proteins were dimeric. Differential scanning calorimetry revealed that cdAE1 and cdDelta54AE1 had similar thermal stabilities with midpoints of transition higher than cdkAE1. cdAE1 and cdDelta54AE1 underwent similar pH-dependent fluorescence changes, while cdkAE1 exhibited a higher intrinsic fluorescence at neutral and acidic pH. Urea denaturation resulted in dequenching of tryptophan fluorescence in cdAE1, while tryptophans in cdkAE1 were already dequenched in the native state. We conclude that the absence of the central beta-strand in cdkAE1 results in a less stable and more open structure than cdAE1. This structural change, in addition to the loss of the acidic amino-terminal region, may account for the altered protein binding properties of kAE1.     

A Substrate Access Tunnel in the Cytosolic Domain Is Not an Essential Feature of the Solute Carrier 4 (SLC4) Family of Bicarbonate Transporters

Anion exchanger 1 (AE1; Band 3; SLC4A1) is the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters that includes chloride/bicarbonate AEs and Na⁺-bicarbonate co-transporters (NBCs). These membrane proteins consist of an amino-terminal cytosolic domain involved in protein interactions and a carboxyl-terminal membrane domain that carries out the transport function. Mutation of a conserved arginine residue (R298S) in the cytosolic domain of NBCe1 (SLC4A4) is linked to proximal renal tubular acidosis and results in impaired transport function, suggesting that the cytosolic domain plays a role in substrate permeation. Introduction of single and double mutations at the equivalent arginine (Arg²⁸³) and at an interacting glutamate (Glu⁸⁵) in the cytosolic domain of human AE1 (cdAE1) had no effect on the cell surface expression or the transport activity of AE1 expressed in HEK-293 cells. In addition, the membrane domain of AE1 (mdAE1) efficiently mediated anion transport. A 2.1-Å resolution crystal structure of cdΔ54AE1 (residues 55–356 of cdAE1) lacking the amino-terminal and carboxyl-terminal disordered regions, produced at physiological pH, revealed an extensive hydrogen-bonded network involving Arg²⁸³ and Glu⁸⁵. Mutations at these residues affected the pH-dependent conformational changes and stability of cdΔ54AE1. As these structural alterations did not impair functional expression of AE1, the cytosolic and membrane domains operate independently. A substrate access tunnel within the cytosolic domain is not present in AE1 and therefore is not an essential feature of the SLC4 family of bicarbonate transporters.     

Luminescence resonance energy transfer investigation of conformational changes in the ligand binding domain of a kainate receptor

The apo state structure of the isolated ligand binding domain of the GluR6 subunit and the conformational changes induced by agonist binding to this protein have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances show that agonist binding induces cleft closure, and the extent of cleft closure is proportional to the extent of activation over a wide range of activations, thus establishing that the cleft closure conformational change is one of the mechanisms by which the agonist mediates receptor activation. The LRET distances also provide insight into the apo state structure, for which there is currently no crystal structure available. The distance change between the glutamate-bound state and the apo state is similar to that observed between the glutamate-bound and antagonist UBP-310-bound form of the GluR5 ligand binding domain, indicating that the cleft for the apo state of the GluR6 ligand binding domain should be similar to the UBP-310-bound form of GluR5. This observation implies that te apo state of GluR6 undergoes a cleft closure of 29-30 degrees upon binding full agonists, one of the largest observed in the glutamate receptor family.     

Proton-mediated Conformational Changes in an Acid-sensing Ion Channel

Acid-sensing ion channels are cation channels activated by external protons and play roles in nociception, synaptic transmission, and the physiopathology of ischemic stroke. Using luminescence resonance energy transfer (LRET), we show that upon proton binding, there is a conformational change that increases LRET efficiency between the probes at the thumb and finger subdomains in the extracellular domain of acid-sensing ion channels. Additionally, we show that this conformational change is lost upon mutating Asp-238, Glu-239, and Asp-260, which line the finger domains, to alanines. Electrophysiological studies showed that the single mutant D260A shifted the EC₅₀ by 0.2 pH units, the double mutant D238A/E239A shifted the EC₅₀ by 2.5 pH units, and the triple mutant D238A/E239A/D260A exhibited no response to protons despite surface expression. The LRET experiments on D238A/E239A/D260A showed no changes in LRET efficiency upon reduction in pH from 8 to 6. The LRET and electrophysiological studies thus suggest that the three carboxylates, two of which are involved in carboxyl/carboxylate interactions, are essential for proton-induced conformational changes in the extracellular domain, which in turn are necessary for receptor activation.     

Role of the buried glutamate in the alpha-helical coiled coil domain of the macrophage scavenger receptor

The macrophage scavenger receptor exhibits a pH-dependent conformational change around the carboxy-terminal half of the alpha-helical coiled coil domain, which has a representative amino acid sequence of a (defgabc)n heptad. We previously demonstrated that a peptide corresponding to this region formed a random coil structure at pH 7 and an alpha-helical coiled coil structure at pH 5 [Suzuki, K., Doi, T., Imanishi, T., Kodama, T., and Tanaka, T. (1997) Biochemistry 36, 15140-15146]. To determine the amino acid responsible for the conformational change, we prepared several peptides in which the acidic amino acids were replaced with neutral amino acids. Analyses of their structures by circular dichroism and sedimentation equilibrium gave the result that the presence of Glu242 at the d position was sufficient to induce the pH-dependent conformational change of the alpha-helical coiled coil domain. Furthermore, we substituted a Glu residue for the Ile residue at the d or a position of a de novo designed peptide (IEKKIEA)4, which forms a highly stable triple-stranded coiled coil. These peptides exhibited a pH-dependent conformational change similar to that of the scavenger receptor. Therefore, we conclude that a buried Glu residue in the hydrophobic core of a triple-stranded coiled coil has the potential to induce the pH-dependent conformational change. This finding makes it possible to elucidate the functions of natural proteins and to create a de novo protein designed to undergo a pH-dependent conformational change.     

Intermonomer flexibility of Ca- and Mg-actin filaments at different pH values.

The fluorescence resonance energy transfer parameter, f, is defined as the efficiency of the energy transfer normalized by the quantum yield of the donor in the presence of acceptor. It is possible to characterize the flexibility of the protein matrix between the appropriate fluorescent probes by monitoring the temperature dependence of f. The intermonomer flexibility of the Ca-actin and Mg-actin filaments was characterized by using this method at pH values of 6.5 and 7.4. The protomers were labeled on Cys374 with donor [N-(((iodoacetyl)amino)ethyl)-5-naphthylamine-1-sulfonate; IAEDANS] or acceptor [5-(iodoacetamido)fluorescein; IAF] molecules. The temperature profile of f suggested that the intermonomer flexibility of actin filaments was larger at pH 7.4 than pH 6.5 in the case of Mg-F-actin while this difference was absent in the case of Ca-F-actin. More rigid intermonomer connection was identified at both pH values between the protomers of Mg-F-actin compared to the Ca-F-actin. The results were further supported by time dependent fluorescence measurements made on IAEDANS and IAF labeled Mg- and Ca-actin filaments at pH 6.5 and 7.4. Our spectroscopic results may suggest that the altered function of muscle following the change of pH within the muscle cells under physiological or pathological conditions might be affected by the modified dynamic properties of the magnesium saturated actin filaments. The change of the intracellular pH does not have an effect on the intermonomer flexibility of the Ca-actin filaments.     

The octapeptide repeats in mammalian prion protein constitute a pH-dependent folding and aggregation site

Structural studies of mammalian prion protein at pH values between 4.5 and 5.5 established that the N-terminal 100 residue domain is flexibly disordered. Here, we show that at pH values between 6.5 and 7.8, i.e. the pH at the cell membrane, the octapeptide repeats in recombinant human prion protein hPrP(23-230) encompassing the highly conserved amino acid sequence PHGGGWGQ are structured. The nuclear magnetic resonance solution structure of the octapeptide repeats at pH 6.2 reveals a new structural motif that causes a reversible pH-dependent PrP oligomerization. Within the aggregation motif the segments HGGGW and GWGQ adopt a loop conformation and a beta-turn-like structure, respectively. Comparison with the crystal structure of HGGGW-Cu(2+) indicates that the binding of copper ions induces a conformational transition that presumably modulates PrP aggregation. The knowledge that the cellular prion protein is immobilized on the cell surface along with our results suggests a functional role of aggregation in endocytosis or homophilic cell adhesion.     

Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils

We describe in molecular detail how disruption of an intermonomer salt bridge (Arg337-Asp352) leads to partial destabilization of the p53 tetramerization domain and a dramatically increased propensity to form amyloid fibrils. At pH 4.0 and 37 degrees C, a p53 tetramerization domain mutant (p53tet-R337H), associated with adrenocortical carcinoma in children, readily formed amyloid fibrils, while the wild-type (p53tet-wt) did not. We characterized these proteins by equilibrium denaturation, 13C(alpha) secondary chemical shifts, (1H)-15N heteronuclear NOEs, and H/D exchange. Although p53tet-R337H was thermodynamically less stable, NMR data indicated that the two proteins had similar secondary structure and molecular dynamics. NMR derived pK(a) values indicated that at low pH the R337H mutation partially disrupted an intermonomer salt bridge. Backbone H/D exchange results showed that for at least a small population of p53tet-R337H molecules disruption of this salt bridge resulted in partial destabilization of the protein. It is proposed that this decrease in p53tet-R337H stability resulted in an increased propensity to form amyloid fibrils.     

Domain 4 of the anthrax protective antigen maintains structure and binding to the host receptor CMG2 at low pH

Domain 4 of the anthrax protective antigen (PA) plays a key role in cellular receptor recognition as well as in pH-dependent pore formation. We present here the 1.95 Å crystal structure of domain 4, which adopts a fold that is identical to that observed in the full-length protein. We have also investigated the structural properties of the isolated domain 4 as a function of pH, as well as the pH-dependence on binding to the von Willebrand factor A domain of capillary morphogenesis protein 2 (CMG2). Our results provide evidence that the isolated domain 4 maintains structure and interactions with CMG2 at pH 5, a pH that is known to cause release of the receptor on conversion of the heptameric prepore (PA₆₃)₇ to a membrane-spanning pore. Our results suggest that receptor release is not driven solely by a pH-induced unfolding of domain 4.     

Luminescence resonance energy transfer measurements in myosin.

Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure distances between the catalytic domain (Cys707) and regulatory light chain domain (Cys108) of the myosin head. An energy transfer efficiency of 21.2 +/- 1.9% is measured in the myosin complex without nucleotide or actin, corresponding to a distance of 73 A, consistent with the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5 +/- 1.0%, indicating a conformational change in myosin that involves a relative rotation and/or translation of Cys707 relative to the light chain domain. Addition of ADP also alters the energy transfer efficiency, likely through a rotation of the probe attached to Cys707. These results demonstrate that LRET is capable of making accurate measurements on the relatively large actomyosin complex, and is capable of detecting conformational changes between the catalytic and light chain domains of myosin.     

pH-dependent structural transition in rabbit skeletal troponin C

Although the crystal structure of troponin C is known (Herzberg, O., and James, M. N. G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., and Wang, B. C. (1985) Science 227, 945-948), its structure in solution, particularly under physiological conditions, has not been established. We examined the conformation of troponin C under a variety of conditions by measuring the distance between sites located in the N- and C-terminal domains using the technique of resonance energy transfer. The donor was the luminescent lanthanide ion Tb3+ bound at the low affinity metal sites in the N-terminal domain. The acceptor was 4-dimethylaminophenylazophenyl-4'-maleimide attached at Cys-98 in the C-terminal domain. The distance between these sites was found to be greater than 5.2 nm at pH 5.0, 2.7 nm at pH 6.8 for uncomplexed troponin C, and 4.1 nm for troponin C complexed with troponin I at pH 6.8. These findings suggest that uncomplexed troponin C undergoes a pH-dependent transition from an elongated conformation, compatible with the crystal structure at acidic pH, to a more compact conformation at neutral pH. When complexed with troponin I, troponin C adopts a conformation of intermediate length compared to the uncomplexed molecule at pH 6.8 and 5.0.     

Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range

A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP(+) or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent pK(a) values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual pK(a) values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP(+) and NADPH forms of domain III, but with a marked difference in pK(a) not only attributable to the charge difference between the substrates. Titrating residues found in loop D/alpha5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent pK(a) values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.     

Calcium coordination and pH dependence of the calcium affinity of ligand-binding repeat CR7 from the LRP. Comparison with related domains from the LRP and the LDL receptor.

We have determined the X-ray crystal structure to 1.8 A resolution of the Ca(2+) complex of complement-like repeat 7 (CR7) from the low-density lipoprotein receptor-related protein (LRP) and characterized its calcium binding properties at pH 7.4 and 5. CR7 occurs in a region of the LRP that binds to the receptor-associated protein, RAP, and other protein ligands in a Ca(2+)-dependent manner. The calcium coordination is identical to that found in LB5 and consists of carboxyls from three conserved aspartates and one conserved glutamate, and the backbone carbonyls of a tryptophan and another aspartate. The overall fold of CR7 is similar to those of CR3 and CR8 from the LRP and LB5 from the LDL receptor, though the low degree of sequence homology of residues not involved in calcium coordination or in disulfide formation results in a distinct pattern of surface residues for each domain, including CR7. The thermodynamic parameters for Ca(2+) binding at both extracellular and endosomal pHs were determined by isothermal titration calorimetry for CR7 and for related complement-like repeats CR3, CR8, and LB5. Although the drop in pH resulted in a reduction in calcium affinity in each case, the changes were very variable in magnitude, being as low as a 2-fold reduction for CR3. This suggests that a pH-dependent change in calcium affinity alone cannot be responsible for the release of bound protein ligands from the LRP at the pH prevailing in the endosome, which in turn requires one or more other pH-dependent effects for regulating protein ligand release.     

Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin

The N-terminal domain of the influenza hemagglutinin (HA) is the only portion of the molecule that inserts deeply into membranes of infected cells to mediate the viral and the host cell membrane fusion. This domain constitutes an autonomous folding unit in the membrane, causes hemolysis of red blood cells and catalyzes lipid exchange between juxtaposed membranes in a pH-dependent manner. Combining NMR structures determined at pHs 7.4 and 5 with EPR distance constraints, we have deduced the structures of the N-terminal domain of HA in the lipid bilayer. At both pHs, the domain is a kinked, predominantly helical amphipathic structure. At the fusogenic pH 5, however, the domain has a sharper bend, an additional 3(10)-helix and a twist, resulting in the repositioning of Glu 15 and Asp 19 relative to that at the nonfusogenic pH 7.4. Rotation of these charged residues out of the membrane plane creates a hydrophobic pocket that allows a deeper insertion of the fusion domain into the core of the lipid bilayer. Such an insertion mode could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.     

Structural origins of pH‐dependent chemical shifts in the B1 domain of protein G

We report chemical shifts for H^N, N, and C′ nuclei in the His‐tagged B1 domain of protein G (GB1) over a range of pH values from pH 2.0 to 9.0, which fit well to standard pH‐dependent equations. We also report a 1.2 Å resolution crystal structure of GB1 at pH 3.0. Comparison of this crystal structure with published crystal structures at higher pHs provides details of the structural changes in GB1 associated with protonation of the carboxylate groups, in particular a conformational change in the C‐terminus of the protein at low pH. An additional change described recently is not seen in the crystal structure because of crystal contacts. We show that the pH‐dependent changes in chemical shifts can be almost entirely understood based on structural changes, thereby providing insight into the relationship between structure and chemical shift. In particular, we describe through‐bond effects extending up to five bonds, affecting N and C′ but not H^N; through‐space effects of carboxylates, which fit well to a simple electric field model; and effects due to conformational change, which have a similar magnitude to many of the direct effects. Finally, we discuss cooperative effects, demonstrating a lack of cooperative unfolding in the helix, and the existence of a β‐sheet “iceberg” extending over three of the four strands. This study therefore extends the application of chemical shifts to understanding protein structure. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

Crystal Structure of Family 14 Polysaccharide Lyase with pH-dependent Modes of Action

The Chlorella virus enzyme vAL-1 (38 kDa), a member of polysaccharide lyase family 14, degrades the Chlorella cell wall by cleaving the glycoside bond of the glucuronate residue (GlcA) through a β-elimination reaction. The enzyme consists of an N-terminal cell wall-attaching domain (11 kDa) and a C-terminal catalytic module (27 kDa). Here, we show the enzyme characteristics of vAL-1, especially its pH-dependent modes of action, and determine the structure of the catalytic module. vAL-1 also exhibited alginate lyase activity at alkaline pH, and truncation of the N-terminal domain increased the lyase activity by 50-fold at pH 7.0. The truncated form vAL-1(S) released di- to hexasaccharides from alginate at pH 7.0, whereas disaccharides were preferentially generated at pH 10.0. This indicates that vAL-1(S) shows two pH-dependent modes of action: endo- and exotypes. The x-ray crystal structure of vAL-1(S) at 1.2 Å resolution showed two antiparallel β-sheets with a deep cleft showing a β-jelly roll fold. The structure of GlcA-bound vAL-1(S) at pH 7.0 and 10.0 was determined: GlcA was found to be bound outside and inside the cleft at pH 7.0 and 10.0, respectively. This suggests that the electric charges at the active site greatly influence the binding mode of substrates and regulate endo/exo activity. Site-directed mutagenesis demonstrated that vAL-1(S) has a specific amino acid arrangement distinct from other alginate lyases crucial for catalysis. This is, to our knowledge, the first study in which the structure of a family 14 polysaccharide lyase with two different modes of action has been determined.     

Coil-to-helix transition and ligand release of Bombyx mori pheromone-binding protein

The transport of hydrophobic insect pheromones through the aqueous medium surrounding their receptors is assisted by pheromone-binding proteins (PBPs). The protein from the silkworm moth Bombyx mori, BmorPBP, exhibits a pH-dependent conformational change postulated to trigger the release of the pheromone bombykol to its receptor. At low pH, an alpha-helix occupies the same binding pocket that houses the pheromone in the BmorPBP-bombykol complex at high pH. We have determined the crystal structure of apo BmorPBP at a resolution of 2.3 angstroms and pH 7.5, which has surprisingly a structure similar to the A-form. These data suggest that BmorPBP undergoes a ligand-dependent conformational change in addition to the previously described pH-dependent conformational change. Analysis of the alpha-helix occupying the binding pocket reveals an amphipathic helix with three acidic residues along one face that are conserved among lepidopteran PBPs and may be involved in a conformational transition of BmorPBP at the receptor membrane.     

Effect of pH on the stability and structure of yeast hexokinase A. Acidic amino acid residues in the cleft region are critical for the opening and the closing of the structure

pH and salts have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. In this work, calorimetry, CD, and fluorescence studies have been carried out to understand the pH-dependent conformational changes of the two-domain protein yeast hexokinase A. In conjunction with the crystal structural data available, the present results have enabled the complete characterization and analysis of the pH-dependent conformational changes of the enzyme that have strong implications in understanding its structure-function relationship. The calorimetric profiles show a single thermal transition in the acidic pH range, whereas two independent transitions were observed in the alkaline pH range, suggesting the structural merger of the domains at the acidic pH. Comparison of the thermal transitions at pH 8.5 studied by different techniques suggests that the first transition corresponds to the smaller domain, and the second transition corresponds to the larger domain. The acid-denatured state of hexokinase A has high secondary structure content with little or no tertiary interactions and binds to the hydrophobic dye 8-anilinonaphthalene-1-sulfonic acid, suggesting that it is a molten globule-like state, whereas the alkali-denatured state is less structured than the acid-denatured state but more structured than the urea-denatured state, suggestive of a premolten globule-like state. Structural analysis using the published hexokinase B structure as well as the hexokinase A structure with the revised amino acid sequence in conjunction with the results obtained by us suggests that the ionization state of the acidic residues at the active site could regulate domain movements that are responsible for the opening and the closure of the cleft between the two domains and in turn affect the structure and function of the enzyme.     

Mechanism of the pH-Induced Conformational Change in the Sensor Domain of the DraK Histidine Kinase via the E83, E105, and E107 Residues

The DraR/DraK two-component system was found to be involved in the differential regulation of antibiotic biosynthesis in a medium-dependent manner; however, its function and signaling and sensing mechanisms remain unclear. Here, we describe the solution structure of the extracellular sensor domain of DraK and suggest a mechanism for the pH-dependent conformational change of the protein. The structure contains a mixed alpha-beta fold, adopting a fold similar to the ubiquitous sensor domain of histidine kinase. A biophysical study demonstrates that the E83, E105, and E107 residues have abnormally high pKa values and that they drive the pH-dependent conformational change for the extracellular sensor domain of DraK. We found that a triple mutant (E83L/E105L/E107A) is pH independent and mimics the low pH structure. An in vivo study showed that DraK is essential for the recovery of the pH of Streptomyces coelicolor growth medium after acid shock. Our findings suggest that the DraR/DraK two-component system plays an important role in the pH regulation of S. coelicolor growth medium. This study provides a foundation for the regulation and the production of secondary metabolites in Streptomyces.     

Zinc- and pH-dependent conformational transition in a putative interdomain linker region of the influenza virus matrix protein M1

The matrix protein M1 of influenza A virus forms a shell beneath the viral envelope and sustains the virion architecture by interacting with other viral components. A structural change of M1 upon acidification of the virion interior in an early stage of virus infection is considered to be a key step to virus uncoating. We examined the structure of a 28-mer peptide (M1Lnk) representing a putative linker region between the N- and C-terminal domains of M1 by using circular dichroism, Raman, and absorption spectroscopy. M1Lnk assumes an alpha-helical structure in a mildly hydrophobic environment irrespective of pH, being consistent with the X-ray crystal structures of an N-terminal fragment of M1 at pH 7 and 4. In the presence of Zn(2+), on the other hand, M1Lnk takes a partially unfolded conformation at neutral pH with a tetrahedral coordination of two Cys residues and two His residues to a Zn(2+) ion in the central part of the peptide. Upon acidification, the peptide releases the Zn(2+) ion and refolds into the alpha-helix-rich structure with a midpoint of transition at pH 5.9. The pH-dependent conformational transition of M1Lnk strongly suggests that the interdomain linker region of M1 also undergoes a pH-dependent unfolding-refolding transition in the presence of Zn(2+). A small but significant portion of the M1 protein is bound to Zn(2+) in the virion, and the Zn(2+)-bound M1 molecule may play a special role in virus uncoating by changing the disposition of the N- and C-terminal domains upon acidification of the virion interior.