Type 2 Ryanodine Receptor Domain A Contains a Unique and Dynamic α-Helix That Transitions to a β-Strand in a Mutant Linked with a Heritable Cardiomyopathy

Ryanodine receptors (RyRs) are large tetrameric calcium (Ca^2 +) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca^2 + sensitivity, propagating sarcoplasmic reticulum luminal Ca^2 + release in the process of excitation–contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1–559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R169Q and delta exon 3 (Δ3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile α-helix, unique to type 2; further, this α2 helix rescues the β-strand lost in RyR2A Δ3 but remains dynamic in the hot-spot loop (HS-loop) P164S, R169Q and R176Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A α2 helix is in close proximity to dense “columns” projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain.     

Alcohol-Induced Conformational Transitions in Ervatamin C. An α-Helix to β-Sheet Switchover

Alcohol-induced conformational transitions of erv C, a highly stable cysteine protease, were followed by CD, fluorescence, and activity. At acidic pH, the addition of different alcohols caused two types of conformational transitions. Increasing the concentration of nonfluorinated alkyl alcohols induced a conformational switch from α-helix to β-sheet. Under these conditions, the protein lost its proteolytic activity and tertiary structure. The switch was a sudden one, observed in 50% methanol, 45% ethanol, and 40% propanol. Under similar conditions of pH and concentration, however, glycerol and TFE enhanced the α-helicity of the protein. Methanol-induced denaturation was observed to occur in two stages; the first is the β-sheet state stabilized at low alcohol concentrations, and the other is the β-sheet state with enhanced ellipticity stabilized at high alcohol concentrations. This β-sheet conformation can be attained from the native as well as 6 M GuHCl-denatured state by addition of methanol and exhibits properties different from the native or unfolded state. This state shows loss of tertiary structure and activity, enhanced nonnative secondary structure, noncooperative temperature unfolding, and higher stability toward denaturants as compared to the native state, which are characteristic of the molten globule-like state or O-state, and thus this state may be functioning as an intermediate in the folding pathway of erv C.     

The Congenital Cataract-Linked A2V Mutation Impairs Tetramer Formation and Promotes Aggregation of βB2-Crystallin

β/γ-Crystallins, the major structural proteins in human lens, are highly conserved in their tertiary structures but distinct in the quaternary structures. The N- and C-terminal extensions have been proposed to play a crucial role in mediating the size of β-crystallin assembly. In this research, we investigated the molecular mechanism underlying the congenital hereditary cataract caused by the recently characterized A2V mutation in βB2-crystallin. Spectroscopic experiments indicated that the mutation did not affect the secondary and tertiary structures of βB2-crystallin. The mutation did not affect the formation of βB2/βA3-crystallin heteromer as well as the stability and folding of the heteromer, suggesting that the mutation might not interfere with the protein interacting network in the lens. However, the tetramerization of βB2-crystallin at high protein concentrations was retarded by the A2V mutation. The mutation slightly decreased the thermal stability and promoted the thermal aggregation of βB2-crystallin. Although it did not influence the stability of βB2-crystallin against denaturation induced by chemical denaturants and UV irradiation, the A2V mutant was more prone to be trapped in the off-pathway aggregation process during kinetic refolding. Our results suggested that the A2V mutation might lead to injury of lens optical properties by decreasing βB2-crystallin stability against heat treatment and by impairing βB2-crystallin assembly into high-order homo-oligomers.     

The H50Q Mutation Induces a 10-fold Decrease in the Solubility of α-Synuclein

The conversion of α-synuclein from its intrinsically disordered monomeric state into the fibrillar cross-β aggregates characteristically present in Lewy bodies is largely unknown. The investigation of α-synuclein variants causative of familial forms of Parkinson disease can provide unique insights into the conditions that promote or inhibit aggregate formation. It has been shown recently that a newly identified pathogenic mutation of α-synuclein, H50Q, aggregates faster than the wild-type. We investigate here its aggregation propensity by using a sequence-based prediction algorithm, NMR chemical shift analysis of secondary structure populations in the monomeric state, and determination of thermodynamic stability of the fibrils. Our data show that the H50Q mutation induces only a small increment in polyproline II structure around the site of the mutation and a slight increase in the overall aggregation propensity. We also find, however, that the H50Q mutation strongly stabilizes α-synuclein fibrils by 5.0 ± 1.0 kJ mol⁻¹, thus increasing the supersaturation of monomeric α-synuclein within the cell, and strongly favors its aggregation process. We further show that wild-type α-synuclein can decelerate the aggregation kinetics of the H50Q variant in a dose-dependent manner when coaggregating with it. These last findings suggest that the precise balance of α-synuclein synthesized from the wild-type and mutant alleles may influence the natural history and heterogeneous clinical phenotype of Parkinson disease.     

Estrogen Receptor β2 Induces Hypoxia Signature of Gene Expression by Stabilizing HIF-1α in Prostate Cancer

The estrogen receptor (ER) β variant ERβ2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ERβ2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ERβ2 interacts with and stabilizes HIF-1α protein in normoxia, thereby inducing a hypoxic gene expression signature. HIF-1α is known to stimulate metastasis by increasing expression of Twist1 and increasing vascularization by directly activating VEGF expression. We found that ERβ2 interacts with HIF-1α and piggybacks to the HIF-1α response element present on the proximal Twist1 and VEGF promoters. These findings suggest that at least part of the oncogenic effects of ERβ2 is mediated by HIF-1α and that targeting of this ERβ2 – HIF-1α interaction may be a strategy to treat prostate cancer.     

A Missense Mutation in CRYBB2 Leads to Progressive Congenital Membranous Cataract by Impacting the Solubility and Function of βB2-Crystallin

Congenital cataract is a major cause of visual impairment and childhood blindness. The solubility and stability of crystallin proteins play critical roles in maintaining the optical transparency of the lens during the life span. Previous studies have shown that approximately 8.3%∼25% of congenital cataracts are inherited, and mutations in crystallins are the most common. In this study, we attempted to identify the genetic defect in a four-generation family affected with congenital cataracts. The congenital cataract phenotype of this four-generation family was identified as membranous cataract by slit-lamp photography. Mutation screening of the candidate genes detected a heterozygous c.465G→C change in the exon6 of the βB2-crystallin gene (CRYBB2) in all family members affected with cataracts, resulting in the substitution of a highly conserved Tryptophan to Cystine (p.W151C). The mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis and found that the transition resulted in the absence of a BslI restriction site in the affected members of the pedigree. The outcome of PolyPhen-2 and SIFT analysis predicted that this W151C mutation would probably damage to the structure and function of βB2-crystallin. Wild type (wt) and W151C mutant βB2-crystallin were expressed in human lens epithelial cells (HLECs), and the fluorescence results showed that Wt-βB2-crystallin was evenly distributed throughout the cells, whereas approximately 34.7% of cells transfected with the W151C mutant βB2-crystallin formed intracellular aggregates. Taken together, these data suggest that the missense mutation in CRYBB2 gene leads to progressive congenital membranous cataract by impacting the solubility and function of βB2-crystallin.     

The residual structure of acid-denatured β2-microglobulin is relevant to an ordered fibril morphology

β₂-Microglobulin (β2m) forms amyloid fibrils in vitro under acidic conditions. Under these conditions, the residual structure of acid-denatured β2m is relevant to seeding and fibril extension processes. Disulfide (SS) bond-oxidized β2m has been shown to form rigid, ordered fibrils, whereas SS bond-reduced β2m forms curvy, less-ordered fibrils. These findings suggest that the presence of an SS bond affects the residual structure of the monomer, which subsequently influences the fibril morphology. To clarify this process, we herein performed NMR experiments. The results obtained revealed that oxidized β2m contained a residual structure throughout the molecule, including the N- and C-termini, whereas the residual structure of the reduced form was localized and other regions had a random coil structure. The range of the residual structure in the oxidized form was wider than that of the fibril core. These results indicate that acid-denatured β2m has variable conformations. Most conformations in the ensemble cannot participate in fibril formation because their core residues are hidden by residual structures. However, when hydrophobic residues are exposed, polypeptides competently form an ordered fibril. This conformational selection phase may be needed for the ordered assembly of amyloid fibrils.     

Cyclooxygenase-2 Enzyme Induces the Expression of the α-2,3-Sialyltransferase-3 (ST3Gal-I) in Breast Cancer

Aberrant glycosylation is a common feature of malignant change. Changes in mucin-type O-linked glycosylation in breast cancer can result in the expression of truncated core 1-based sialylated glycans rather than the core 2-based glycans observed in normal mammary epithelium cells. This has been shown, in part, to be due to changes in the expression of glycosyltransferases, including the up-regulation of some sialyltransferases. Using the breast cancer cell line T47D, we have shown that PGE2, one of the final products of the cyclooxygenase-2 (COX-2) pathway, can induce the mRNA expression of the sialyltransferase α-2,3-sialyltransferase-3 (ST3Gal-I), resulting in increased sialyltransferase activity, demonstrated by a reduction in PNA lectin staining. Induction of COX-2 in the MDA-MB-231 breast cancer cell line also results in the increased expression of ST3Gal-I, leading to increased sialylation of the substrate of ST3Gal-I, core 1 Galβ1,3GalNAc. This effect on sialylation could be reversed by the selective COX-2 inhibitor celecoxib. The use of siRNA to knock down COX-2 and overexpression of COX-2 in MDA-MD-231 cells confirmed the involvement of COX-2 in the up-regulation of ST3Gal-I. Moreover, analysis of the expression of ST3Gal-I and COX-2 by 74 primary breast cancers showed a significant correlation between the two enzymes. COX-2 expression has been associated with a number of tumors, including breast cancer, where its expression is associated with poor prognoses. Thus, these results suggest the intriguing possibility that some of the malignant characteristics associated with COX-2 expression may be via the influence that COX-2 exerts on the glycosylation of tumor cells.     

The α1A-Adrenoceptor couples to Ca2+ mobilization in mouse peritoneal macrophages

Activation of the α₁-adrenoceptor (α₁-AR) in mouse peritoneal macrophages induces a Ca²⁺-dependent K⁺ current via release of Ca²⁺ from internal stores [Hara et al. (1991) Pflügers Arch 419: 371–379]. In order to characterize the α₁-AR subtype in macrophages, we examined potencies of the α₁-AR subtype-selective antagonists against epinephrine-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i). The K_i values for the α₁-AR in macrophages of WB4101, 5-methylurapidil and phentolamine were 0.13 nM, 0.15 nM and 7.0 nM, respectively. (+)-Niguldipine (10 nM) completely blocked the response. Treatment with chloroethylclonidine (CEC) did not abolish the response. These pharmacological properties of the α₁-AR in macrophages agree quite well with those of the α_1A subtype.     

The α2L111R,N122D Isoform of the Na,K-ATPase Expressed in HeLa Cells Does Not Undergo an Adipocyte-like Increase in Activity in Response to Insulin

In the rat adipocyte, insulin increases potassium uptake by a preferential activation of the α2 isoform of the Na,K-ATPase. The question under consideration here is whether expression of the α2 isoform is sufficient to replicate its differential activation by insulin. Accordingly, we compared the effect of insulin on the activity of the ouabain resistant rat α1 and α2RD (α2L111R,N122D) isoforms in HeLa cells. In HeLa cells, in contrast to the rat adipocyte, insulin produces an increase of equal magnitude in the rate of⁸⁶Rb⁺/K⁺uptake by the ouabain resistant rat α1 and rat α2RD subunits. We conclude that the mechanism of insulin activation of the α2RD isoform in HeLa cells differs from that of the wild type α2 isoform in the rat adipocyte.     

Key Residues in the Nicotinic Acetylcholine Receptor β2 Subunit Contribute to α-Conotoxin LvIA Binding

α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val¹¹¹ is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr⁵⁹ and Phe¹¹⁹ of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.     

Soluble guanylate cyclase isoenzymes: The expression of α1, α2, β1, and β2 subunits in the benign and malignant breast tumors

Soluble guanylate cyclase (sGC) encompasses α and β subunits. This study examined the expression of α1, α2, β1, and β2 subunits in the malignant and benign breast tumors using the Western blot analysis. Both benign and malignant tumors showed a significantly higher expression of the α1 subunit in comparison with normal tissues (p < 0.0001). In contrast, the expression of α2 and β2 sGC were significantly lower in these tumors than normal tissues (p < .0015 and p < .001, p < .007 and p < .0001, respectively). The expression level of α1 sGC was significantly correlated with ER + PR+ (p < .0001). A significant correlation was also detected for sGC-α1 and -α2 expression with c-erbB2-negative status (p < .01). However, the expression level of sGC was not associated with tumor stage, tumor grade, or other clinicopathological features. In conclusion, as the expression of α1 sGC is upregulated and α2 and β2 sGC are downregulated in malignant breast tumors. Variations in the expression of sGC isoenzymes may be suggested as an indicator to confirm the enzyme antitumor activity.     

Epigenetic activation of α4, β2 and β6 integrins involved in cell migration in trichostatin A-treated Hep3B cells

Summary The epigenetic modulation by histone deacetylase (HDAC) inhibitors including trichostatin A (TSA) has been known to block cell proliferation, induce apoptosis and inhibit cell migration in human cancer cells that represents the potential therapeutic agents for cancers and fibrosis. However, more than 55% of Hep3B cells remained alive after our initial study of 100 nM TSA treatment. To further study the epigenetic modulation and the biological function of newly activated genes by HDAC inhibitor involved in HCC progression and metastasis, we profiled 23 integrin genes including 15α and 8β in TSA-treated Hep3B cells. Six integrins including three down-regulated α6, α10, β8 and three significant up-regulated α4, β2, β6 integrins were revealed after semi-quantitative RT-PCR. To confirm the epigenetic modulation and explore their biological functions, we selected the three significantly up-regulated integrins for confirmation of protein up-regulation, hyperacetylated-histones by ChIP assays, and functional inhibition by specific neutralizing antibodies of integrins. Our results indicated that epigenetic modulation in TSA-treated Hep3B cells up-regulated new integrins including α4, β2 and β6 and reduced migration activities by specific neutralizing antibodies to 61.3%, 42.4% and 34.5%, respectively. Our novel findings provided a better understanding of the epigenetic modulation of integrins and suggested that targeting the epigenetic up-regulated integrins to abrogate the migration activity might be a promising strategy to prevent HCC progression.     

The Novel S527F Mutation in the Integrin ��3 Chain Induces a High Affinity ��IIb��3 Receptor by Hindering Adoption of the Bent Conformation

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor αIIbβ3 in a patient with an ill defined platelet disorder: one in the β3 gene (S527F) and two in the αIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant αIIbβ3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the β3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated αIIbβ3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of αIIbβ3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the αIIbβ3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe⁵²⁷ in β3 and the calf-1 domain in αIIb and by decreased flexibility between I-EGF2 and I-EGF3.The platelet receptor αIIbβ3 belongs to the family of integrin receptors that consist of noncovalently linked α/β-heterodimers. They are cell-surface receptors that play a role in cell-cell and cell-matrix interactions. Under resting conditions, integrin receptors adopt the low affinity conformation and do not interact with their ligands. Inside-out signaling turns the receptor into a high affinity conformation capable of ligand binding. Ligand binding itself induces additional conformational changes resulting in exposure of neoantigenic sites called ligand-induced binding sites (LIBS)³ and generates in turn outside-in signaling, which triggers a range of downstream signals (1, 2).Integrin αIIbβ3 is expressed on platelets and megakaryocytes. In flowing blood under resting conditions, αIIbβ3 does not interact with its ligand fibrinogen. When a blood vessel is damaged, platelets adhere at sites of vascular injury and become activated. As a consequence, αIIbβ3 adopts the high affinity conformation and binds fibrinogen. This results in platelet aggregation and thrombus formation, which eventually will stop the bleeding (3).The topology of integrins comprises an extracellular, globular, N-terminal ligand-binding head domain (the β-propeller domain in the αIIb chain and the βI domain in the β3 chain) standing on two long legs or stalks (consisting of thigh, calf-1, and calf-2 domains in the αIIb chain and hybrid, plexin/semaphorin/integrin (PSI), four integrin endothelial growth factor-like (I-EGF), and β-tail domains in the β3 chain), followed by transmembrane and cytoplasmic domains (1, 2). X-ray crystal structures of the extracellular domain of non-activated αVβ3 revealed that the legs are severely bent, putting the head domain next to the membrane-proximal portions of the legs (4, 5). The bending occurs between I-EGF1 and I-EGF2 in the β-subunit and between the thigh and calf-1 domains in the α-subunit. This bent conformation represents the low affinity state of the receptor. The high affinity state of the receptor is induced by activation and is associated with a large-scale conformational rearrangement in which the integrin extends with a switchblade-like motion (2). Recently, the crystal structure of the entire extracellular domain of αIIbβ3 in its low affinity conformation was resolved and revealed that this integrin also adopts the bent conformation under resting conditions (6). Structural rearrangements in αIIbβ3 between the bent and extended conformations are similar to what has been reported for other integrins (7).We report here that the S527F mutation in the I-EGF3 region of the β3 polypeptide chain of the αIIbβ3 receptor induces a constitutively active receptor adopting an extended high affinity conformation. This was evidenced by spontaneous PAC-1, fibrinogen, and anti-LIBS antibody binding. These data were further corroborated by modeling the replacement of Ser⁵²⁷ with Phe in the crystal structure of the extracellular domain of αIIbβ3. In this model, the S527F mutation decreases the flexibility of I-EGF3 and appears to prevent movement of the lower β-leg into the cleft between the upper β-leg and the lower α-leg. As a consequence, formation of the bent conformation of the non-activated receptor is hampered.     

Myoglobin-H2O2 catalyzes the oxidation of β-ketoacids to α-dicarbonyls: mechanism and implications in ketosis

Acetoacetate (AA) and 2-methylacetoacetate (MAA) are accumulated in metabolic disorders such as diabetes and isoleucinemia. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by myoglobin (Mb)/H₂O₂. We propose a chemiluminescent route involving a dioxetanone intermediate whose thermolysis yields triplet α-dicarbonyl species (methylglyoxal and diacetyl). The observed ultraweak chemiluminescence increased linearly on raising the concentration of either Mb (10-500 μM) or AA (10-100 mM). Oxygen uptake studies revealed that MAA is almost a 100-fold more reactive than AA. EPR spin-trapping studies with MNP/MAA revealed the intermediacy of an α-carbon-centered radical and acetyl radical. The latter radical, probably derived from triplet diacetyl, is totally suppressed by sorbate, a well-known quencher of triplet carbonyls. Furthermore, an EPR signal assignable to MNP-AA^• adduct was observed and confirmed by isotope effects. Oxygen consumption and α-dicarbonyl yield were shown to be dependent on AA or MAA concentrations (1-50 mM) and on H₂O₂ or tert-butOOH added to the Mb-containing reaction mixtures. That ferrylMb is involved in a peroxidase cycle acting on the substrates is suggested by the reaction pH profiles and immunospin-trapping experiments. The generation of radicals and triplet dicarbonyl products by Mb/H₂O₂/β-ketoacids may contribute to the adverse health effects of ketogenic unbalance.     

An Activating Mutation Reveals a Second Binding Mode of the Integrin α2 I Domain to the GFOGER Motif in Collagens

The GFOGER motif in collagens (O denotes hydroxyproline) represents a high-affinity binding site for all collagen-binding integrins. Other GxOGER motifs require integrin activation for maximal binding. The E318W mutant of the integrin α2β1 I domain displays a relaxed collagen specificity, typical of an active state. E318W binds more strongly than the wild-type α2 I domain to GMOGER, and forms a 2:1 complex with a homotrimeric, collagen-like, GFOGER peptide. Crystal structure analysis of this complex reveals two E318W I domains, A and B, bound to a single triple helix. The E318W I domains are virtually identical to the collagen-bound wild-type I domain, suggesting that the E318W mutation activates the I domain by destabilising the unligated conformation. E318W I domain A interacts with two collagen chains similarly to wild-type I domain (high-affinity mode). E318W I domain B makes favourable interactions with only one collagen chain (low-affinity mode). This observation suggests that single GxOGER motifs in the heterotrimeric collagens V and IX may support binding of activated integrins.     

Blockage of Conformational Changes of Heat Shock Protein gp96 on Cell Membrane by a α-Helix Peptide Inhibits HER2 Dimerization and Signaling in Breast Cancer

Cell membrane translocation of heat shock protein gp96 from the endoplasmic reticulum has been observed in multiple tumors and is associated with tumor malignancy. However, the cancer-intrinsic function and the related mechanism of cell membrane gp96 as a pro-oncogenic chaperone remain further elucidated. In this study, we found that inhibition of gp96 intramolecular conformational changes by a single α-helix peptide p37 dramatically increased its binding to HER2, whereas decreased HER2 dimerization, phosphorylation and downstream signaling. Targeting cell membrane gp96 promoted HER2 ubiquitination and subsequent lysosomal degradation, which led to decreased cell growth and increased apoptosis, and inhibited tumor growth in vivo. We also demonstrate that gp96 inhibitory peptide p37 synergized with trastuzumab to suppress cell growth and induce apoptosis. Our work demonstrates that blocking gp96 conformational changes directs HER2 for cellular degradation, and represents a new therapeutic strategy for inhibiting HER2 signaling in cancer.