Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson–Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.

Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking

Subunits a and c of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by the Escherichia coli F1Fo ATP synthase. Optimizing mutations in subunit a at residues A217, I221, and L224 improves the partial function of the cA24D/cD61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a. After treating the membrane vesicles of these mutants with Cu(1,10-phenanthroline)2SO4 at 0°, 10°, or 20°C, strong a–c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane. Lesser a–c dimer formation was observed in nine other double mutants after treatment at 20°C in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunit c and helix-4 of subunit a in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunit c and helix-4 of subunit a pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.

Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells

DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif. The Cys-30–Cys-33 disulfide is donated to a pair of cysteines on the target proteins. Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo. DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning. It is not known, however, what might provide the overall oxidizing power to the DsbA–DsbB disulfide bond formation system. We now report that E. coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone/menaquinone deprivation. Disulfide bond formation of β-lactamase was impaired under these conditions. Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA. This is followed by reduction of the bulk of DsbA molecules. These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB. It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

An in vivo pathway for disulfide bond isomerization in Escherichia coli

Biochemical studies have shown that the periplasmic protein disulfide oxidoreductase DsbC can isomerize aberrant disulfide bonds. Here we present the first evidence for an in vivo role of DsbC in disulfide bond isomerization. Furthermore, our data suggest that the enzymes DsbA and DsbC play distinct roles in the cell in disulfide bond formation and isomerization, respectively. We have shown that mutants in dsbC display a defect in disulfide bond formation specific for proteins with multiple disulfide bonds. The defect can be complemented by the addition of reduced dithiothreitol to the medium, suggesting that absence of DsbC results in accumulation of misoxidized proteins. Mutations in the dipZ and trxA genes have similar phenotypes. We propose that DipZ, a cytoplasmic membrane protein with a thioredoxin-like domain, and thioredoxin, the product of the trxA gene, are components of a pathway for maintaining DsbC active as a protein disulfide bond isomerase.

Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm

Under physiological conditions, the Escherichia coli cytoplasm is maintained in a reduced state that strongly disfavors the formation of stable disulfide bonds in proteins. However, mutants in which the reduction of both thioredoxins and glutathione is impaired (trxB gor mutants) accumulate oxidized, enzymatically active alkaline phosphatase in the cytoplasm. These mutants grow very poorly in the absence of an exogenous reductant and accumulate extragenic suppressors at a high frequency. One such suppressor strain, FA113, grows almost as rapidly as the wild type in the absence of reductant, exhibits slightly faster kinetics of disulfide bond formation, and has fully induced activity of the transcriptional activator, OxyR. FA113 gave substantially higher yields of properly oxidized proteins compared with wild-type or trxB mutant strains. For polypeptides with very complex patterns of disulfide bonds, such as vtPA and the full-length tPA, the amount of active protein was further enhanced up to 15-fold by co-expression of TrxA (thioredoxin 1) mutants with different redox potentials, or 20-fold by the protein disulfide isomerase, DsbC. Remarkably, higher yields of oxidized, biologically active proteins were obtained by expression in the cytoplasm of E. coli FA113 compared with what could be achieved via secretion into the periplasm of a wild-type strain, even under optimized conditions. These results demonstrate that the cytoplasm can be rendered sufficiently oxidizing to allow efficient formation of native disulfide bonds without compromising cell viability.

Roles of a conserved arginine residue of DsbB in linking protein disulfide-bond-formation pathway to the respiratory chain of Escherichia coli

The active-site cysteines of DsbA, the periplasmic disulfide-bond-forming enzyme of Escherichia coli, are kept oxidized by the cytoplasmic membrane protein DsbB. DsbB, in turn, is oxidized by two kinds of quinones (ubiquinone for aerobic and menaquinone for anaerobic growth) in the electron-transport chain. We describe the isolation of dsbB missense mutations that change a highly conserved arginine residue at position 48 to histidine or cysteine. In these mutants, DsbB functions reasonably well aerobically but poorly anaerobically. Consistent with this conditional phenotype, purified R48H exhibits very low activity with menaquinone and an apparent Michaelis constant (Km) for ubiquinone seven times greater than that of the wild-type DsbB, while keeping an apparent Km for DsbA similar to that of wild-type enzyme. From these results, we propose that this highly conserved arginine residue of DsbB plays an important role in the catalysis of disulfide bond formation through its role in the interaction of DsbB with quinones.

Forced unfolding modulated by disulfide bonds in the Ig domains of a cell adhesion molecule

Cell adhesion molecules (CAMs) mediate cell attachment and stress transfer through extracellular domains. Here we forcibly unfold the Ig domains of a prototypical Ig superfamily CAM that contains intradomain disulfide bonds. The Ig domains of all such CAMs have conformations homologous to cadherin extracellular domains, titin Ig-type domains, and fibronectin type-III (FNIII) domains. Atomic force microscopy has been used to extend the five Ig domains of Mel-CAM (melanoma CAM)—a protein that is overexpressed in metastatic melanomas—under conditions where the disulfide bonds were either left intact or disrupted through reduction. Under physiological conditions where intradomain disulfide bonds are intact, partial unfolding was observed at forces far smaller than those reported previously for either titin's Ig-type domains or tenascin's FNIII domains. This partial unfolding under low force may be an important mechanism for imparting elasticity to cell–cell contacts, as well as a regulatory mechanism for adhesive interactions. Under reducing conditions, Mel-CAM's Ig domains were found to fully unfold through a partially folded state and at slightly higher forces. The results suggest that, in divergent evolution of all such domains, stabilization imparted by disulfide bonds relaxes requirements for strong, noncovalent, folded-state interactions.

An isolated, surface-expressed I domain of the integrin αLβ2 is sufficient for strong adhesive function when locked in the open conformation with a disulfide bond

We introduced disulfide bonds to lock the integrin αLβ2 I domain in predicted open, ligand binding or closed, nonbinding conformations. Transfectants expressing αLβ2 heterodimers containing locked-open but not locked-closed or wild-type I domains constitutively adhered to intercellular adhesion molecule-1 (ICAM-1) substrates. Locking the I domain closed abolished constitutive and activatable adhesion. The isolated locked-open I domain bound as well as the activated αLβ2 heterodimer, and binding was abolished by reduction of the disulfide. Lovastatin, which binds under the conformationally mobile C-terminal α-helix of the I domain, inhibited binding to ICAM-1 by αLβ2 with wild-type, but not locked-open I domains. These data establish the importance of conformational change in the αL I domain for adhesive function and show that this domain is sufficient for full adhesive activity.

Locking in alternate conformations of the integrin αLβ2 I domain with disulfide bonds reveals functional relationships among integrin domains

We used integrin αLβ2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins. mAbs to the αL I domain and β2 I-like domain inhibit adhesion of wild-type αLβ2 to intercellular adhesion molecule-1. However, with αLβ2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and (ii) that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, αLβ2 containing a locked open I domain was completely resistant to inhibition by mAbs to the β2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type αLβ2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by Mn2+, as measured with mAb m24, which we map here to the β2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or Mn2+-induced exposure of the KIM127 epitope in the β2 stalk region. Furthermore, locked open I domains, in αLβ2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in Mg2+ and Mn2+. These results suggest that Mn2+ activates αLβ2 by binding to a site other than the I domain, most likely the I-like domain of β2.

A role for intermolecular disulfide bonds in prion diseases?

The key event in prion diseases seems to be the conversion of the prion protein PrP from its normal cellular isoform (PrPC) to an aberrant “scrapie” isoform (PrPSc). Earlier studies have detected no covalent modification in the scrapie isoform and have concluded that the PrPC → PrPSc conversion is a purely conformational transition involving no chemical reactions. However, a reexamination of the available biochemical data suggests that the PrPC → PrPSc conversion also involves a covalent reaction of the (sole) intramolecular disulfide bond of PrPC. Specifically, the data are consistent with the hypothesis that infectious prions are composed of PrPSc polymers linked by intermolecular disulfide bonds. Thus, the PrPC → PrPSc conversion may involve not only a conformational transition but also a thiol/disulfide exchange reaction between the terminal thiolate of such a PrPSc polymer and the disulfide bond of a PrPC monomer. This hypothesis seems to account for several unusual features of prion diseases.

Structure and function in rhodopsin: Mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants

Retinitis pigmentosa (RP) point mutations in both the intradiscal (ID) and transmembrane domains of rhodopsin cause partial or complete misfolding of rhodopsin, resulting in loss of 11-cis-retinal binding. Previous work has shown that misfolding is caused by the formation of a disulfide bond in the ID domain different from the native Cys-110–Cys-187 disulfide bond in native rhodopsin. Here we report on direct identification of the abnormal disulfide bond in misfolded RP mutants in the transmembrane domain by mass spectrometric analysis. This disulfide bond is between Cys-185 and Cys-187, the same as previously identified in misfolded RP mutations in the ID domain. The strategy described here should be generally applicable to identification of disulfide bonds in other integral membrane proteins.

Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activity in vivo

The integrin αLβ2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the αM and α2 subunits has been crystallized in both open and closed conformations; however, the αL I domain has been crystallized in only the closed conformation. We hypothesized that the αL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the αL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg2+. Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The kon, koff, and KD values for the locked open I domain were within 1.5-fold of values previously determined for the αLβ2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized αLβ2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.

Detecting the conformational change of transmembrane signaling in a bacterial chemoreceptor by measuring effects on disulfide cross-linking in vivo.

Transmembrane signaling by bacterial chemoreceptors is thought to involve relative movement among the four transmembrane helices of the homodimer. We assayed that movement by measuring effects of ligand occupancy on rates of oxidative cross-linking between cysteines introduced into neighboring helices of the transmembrane domain of chemoreceptor Trg from Escherichia coli. Measurements were done on chemoreceptors in their native environment, intact cells that were motile and chemotactically responsive. Receptor occupancy did not appear to cause drastic rearrangement of the four-helix structure since, among 67 cysteine pairs tested, the same 19 exhibited oxidative cross-linking in the presence or absence of saturating chemoattractant. However, occupancy did cause subtle changes that were detected as effects on rates of cross-linking. Among the seven disulfides appropriate for measurements of initial rates of formation, ligand occupancy had significant and different effects on all three cross-links that connected the two helices within a subunit but had minimal effects on the four that spanned the packing interface between subunits. This constitutes direct evidence that the conformational change of transmembrane signaling involves significant movement within a subunit and minimal movement between subunits, a pattern deduced from several previous studies and now documented directly. Among possible modes of movement between the two helices of a subunit, axial sliding of one helix relative to the other was the conformational change that best accounted for the observed effects on cross-linking.

The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern.

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.

Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex.

Agrobacterium tumefaciens VirB proteins are essential for gene transfer from bacteria to plants. These proteins are postulated to form a transport pore to allow transfer of the T-strand DNA intermediate. To study the function of the VirB proteins in DNA transfer, we developed an expression system in A. tumefaciens. Analysis of one VirB protein, VirB9, by Western blot assays showed that under nonreducing conditions VirB9, when expressed alone, migrates as a approximately 31-kDa band but that it migrates as a approximately 36-kDa band when expressed with all other VirB proteins. The 36-kDa band is converted to the 31-kDa band by the reducing agent 2-mercaptoethanol. Using strains that contain a deletion in a defined virB gene and strains that express specific VirB proteins, we demonstrate that the 36-kDa band is composed of VirB9 and VirB7 that are linked to each other by a disulfide bond. Mutational studies demonstrate that cysteine residues at positions 24 of VirB7 and 262 of VirB9 participate in the formation of this complex.