X-ray structure of a mammalian stearoyl-CoA desaturase.

Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl-CoAs. Because the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologues (SCD1 and SCD5), while mice have four (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity. Thus, SCD1 is a pharmacological target in the treatment of obesity, diabetes and other metabolic diseases. SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyses the formation of a cis-double bond between the ninth and tenth carbons of stearoyl- or palmitoyl-CoA. The reaction requires molecular oxygen, which is activated by a di-iron centre, and cytochrome b5, which regenerates the di-iron centre. To understand better the structural basis of these characteristics of SCD function, here we crystallize and solve the structure of mouse SCD1 bound to stearoyl-CoA at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain, and a plausible pathway for lateral substrate access and product egress. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel and the conformation of the bound acyl chain provide a structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The dimetal centre is coordinated by a unique spacial arrangement of nine conserved histidine residues that implies a potentially novel mechanism for oxygen activation. The structure also illustrates a possible route for electron transfer from cytochrome b5 to the di-iron centre.

Structure of a membrane-embedded prenyltransferase homologous to UBIAD1.

Membrane-embedded prenyltransferases from the UbiA family catalyze the Mg2+-dependent transfer of a hydrophobic polyprenyl chain onto a variety of acceptor molecules and are involved in the synthesis of molecules that mediate electron transport, including Vitamin K and Coenzyme Q. In humans, missense mutations to the protein UbiA prenyltransferase domain-containing 1 (UBIAD1) are responsible for Schnyder crystalline corneal dystrophy, which is a genetic disease that causes blindness. Mechanistic understanding of this family of enzymes has been hampered by a lack of three-dimensional structures. We have solved structures of a UBIAD1 homolog from Archaeoglobus fulgidus, AfUbiA, in an unliganded form and bound to Mg2+ and two different isoprenyl diphosphates. Functional assays on MenA, a UbiA family member from E. coli, verified the importance of residues involved in Mg2+ and substrate binding. The structural and functional studies led us to propose a mechanism for the prenyl transfer reaction. Disease-causing mutations in UBIAD1 are clustered around the active site in AfUbiA, suggesting the mechanism of catalysis is conserved between the two homologs.

Human polymerase theta helicase positions DNA microhomologies for double-strand break repair.

DNA double-strand breaks occur in all human cells on a daily basis and must be repaired with high fidelity to minimize genomic instability1. Deficiencies in high-fidelity DNA repair by homologous recombination lead to dependence on DNA polymerase theta, which identifies DNA microhomologies in 3' single-stranded DNA overhangs and anneals them to initiate error-prone double-strand break repair. The resulting genomic instability is associated with numerous cancers, thereby making this polymerase an attractive therapeutic target2,3. However, despite the biomedical importance of polymerase theta, the molecular details of how it initiates DNA break repair remain unclear4,5. Here we present cryo-electron microscopy structures of the polymerase theta helicase domain bound to microhomology-containing DNA, revealing DNA-induced rearrangements of the helicase that enable DNA repair. Our structures show that DNA-bound helicase dimers facilitate a microhomology search that positions 3' single-stranded DNA ends in proximity to align complementary base pairs and anneal DNA microhomology. We define the molecular determinants that enable the polymerase theta helicase domain to identify and pair DNA microhomologies to initiate mutagenic DNA repair, providing mechanistic insights into therapeutic targeting of these interactions.

Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation.

Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR's TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR's TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR's gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.

Structural basis for pharmacological modulation of the TRPC6 channel.

Transient receptor potential canonical (TRPC) proteins form nonselective cation channels that play physiological roles in a wide variety of cells. Despite growing evidence supporting the therapeutic potential of TRPC6 inhibition in treating pathological cardiac and renal conditions, mechanistic understanding of TRPC6 function and modulation remains obscure. Here we report cryo-EM structures of TRPC6 in both antagonist-bound and agonist-bound states. The structures reveal two novel recognition sites for the small-molecule modulators corroborated by mutagenesis data. The antagonist binds to a cytoplasm-facing pocket formed by S1-S4 and the TRP helix, whereas the agonist wedges at the subunit interface between S6 and the pore helix. Conformational changes upon ligand binding illuminate a mechanistic rationale for understanding TRPC6 modulation. Furthermore, structural and mutagenesis analyses suggest several disease-related mutations enhance channel activity by disrupting interfacial interactions. Our results provide principles of drug action that may facilitate future design of small molecules to ameliorate TRPC6-mediated diseases.

Correction: Cryo-EM Structure of HER2-trastuzumab-pertuzumab complex.

[This corrects the article DOI: 10.1371/journal.pone.0216095.].

Cryo-EM Structure of HER2-trastuzumab-pertuzumab complex.

Trastuzumab and pertuzumab are monoclonal antibodies that bind to distinct subdomains of the extracellular domain of human epidermal growth factor receptor 2 (HER2). Adding these monoclonal antibodies to the treatment regimen of HER2-positive breast cancer has changed the paradigm for treatment in that form of cancer. Synergistic activity has been observed with the combination of these two antibodies leading to hypotheses regarding the mechanism(s) and to the development of bispecific antibodies to maximize the clinical effect further. Although the individual crystal structures of HER2-trastuzumab and HER2-pertuzumab revealed the distinct binding sites and provided the structural basis for their anti-tumor activities, detailed structural information on the HER2-trastuzumab-pertuzumab complex has been elusive. Here we present the cryo-EM structure of HER2-trastuzumab-pertuzumab at 4.36 Å resolution. Comparison with the binary complexes reveals no cooperative interaction between trastuzumab and pertuzumab, and provides key insights into the design of novel, high-avidity bispecific molecules with potentially greater clinical efficacy.

Comparison of Pulmonary Artery Pressure Measurement With Doppler Echocardiography or With Right Heart Catheterization in Patients With Congenital Heart Disease.

Background: Doppler echocardiography (D-ECHO) is a commonly used imaging tool for both diagnosis and follow-up examination of congenital heart disease (CHD). The goal of this study is to evaluate the accuracy of D-ECHO as used to measure an estimate sPAP in pediatric patients with CHD. Methods: A prospective study in 397 pediatric patients with CHD has been carried out to compare estimate sPAP measured with D-ECHO to that measured with right heart catheterization (RHC). Pearson correlation analyses were used to calculate the correlation coefficients between RHC and D-ECHO. Bland-Altman analyses were carried out to assess the agreement between the two methods. Results: Our data have demonstrated a significant underestimation of sPAP by D-ECHO compared to that by RHC. A strong correlation (r = 0.957, p < 0.01) was found between sPAP (36.1 ± 14.9 mmHg) and RVSP (36.0 ± 14.5 mmHg) measured with RHC. However, a relatively weak correlation (r = 0.219, p < 0.01) was observed between sPAP (36.1 ± 14.9 mmHg) measured during RHC and sPAP (28.7 ± 9.7 mmHg) as estimated using D-ECHO. The Bland-Altman analysis demonstrated that the bias for D-ECHO sPAP estimates was 6.6 mmHg with 95% limits of agreement ranging from -23.6 to 36.8 mmHg. A total of 57.5% of D-ECHO measurements were found to be accurate, with accuracy predefined as 95% of agreement within ±10 mmHg for sPAP estimates. Conclusions: sPAP measured with D-ECHO may be underestimated in pediatric patients with CHD.

Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7).

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel.

Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation.

Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an alpha helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore.

[Mutation analysis of Cited2 in patients with congenital heart disease].

OBJECTIVE: To explore mutation of Cited2 gene coding strand in Chinese patients with congenital heart disease (CHD). METHODS: DNA was extracted from the blood samples of 120 nonhomologous and various CHD patients and 100 healthy children. The sequence of coding regions of Cited2 was amplified by PCR and compared to those in the GeneBank after sequencing to identify the mutations. The family of the samples who have Cited2 mutations were investigated as well. Clustal W software was applied for conservative analysis of the altered amino acids. RESULTS: Three new mutations of Cited2 coding strand were found in 4 CHD patients. Two point mutations were first identified respectively in two patients, one patient with mirror image dextrocardia and tetralogy of Fallot (c.550 G > A), another with aortic stenosis (c.574 A > G). Apart from this, the same deletion (c.573-578del6) was first detected in another two patients, one with ventricular septal defect and atrial septal defect, the other with aortic stenosis and pulmonary stenosis. All the mutations resulted in the protein changes (p.Gly184Ser; p.Ser192Gly; p.Ser192fs). None of these changes were detected in the control group. CONCLUSION: This study showed that there are 3 brand-new gene mutations as demonstrated by sequencing of Cited2 gene in Chinese CHD patients with a broad phenotype spectrum. Serine-glycine rich junction (SGJ) is considered as the mutation hot spot. Cited2 mutations may be one of the causes of the development of CHD in human.

[Preliminary study on the effect of carvedilol on children with primary endocardial fibroelastosis].

OBJECTIVE: Endocardial fibroelastosis (EFE), a common pediatric cardiovascular disease, often results in chronic heart failure (CHF) and death. Clinical trials have shown that the regimen of combining beta-adrenoreceptor blocker with traditional medicines against CHF can improve left ventricular function and prevent the ventricle from remodeling in patients with CHF. The present study aimed to observe the effect of carvedilol on concentration of plasma brain-type natriuretic peptide (BNP), and safety in children with EFE. METHODS: Twenty-one children with EFE were randomly divided into two groups: (1) treated with traditional regimen (digoxin, prednisone and/or diuretics) (n = 10); (2) treated with carvedilol plus traditional regimen (n = 11). Measurement of plasma concentration of BNP by ELISA, cardiac function by ultrasound were performed before and after 6 months of treatment. The changes in clinical symptom, heart rate, heart function, side effect and maximal tolerance dose after treatment with carvedilol were observed. RESULTS: Plasma concentration of BNP was much higher in the group of patients with EFE [(865 +/- 702) ng/L] than that of control group [(154 +/- 78) ng/L] (P < 0.01), and there was a positive correlation between plasma concentration of BNP and cardiac function classification, and cardiac function grades II, III, and IV corresponded to plasma concentration of BNP (286 +/- 125) ng/L, (437 +/- 386) ng/L, (1673 +/- 859) ng/L respectively in children with EFE. Compared with the group treated with traditional medicines, plasma concentration of BNP [(403 +/- 216) ng/L vs. (219 +/- 87) ng/L] significantly decreased, the clinical symptom was significantly improved, cardio-thoracic ratio (CTR) (0.60 +/- 0.05 vs. 0.54 +/- 0.06) (P < 0.05) and heart rate [(115 +/- 20) bpm vs. (90 +/- 14) bpm] (P < 0.01) decreased, ejection fraction (EF) (46.6% +/- 13.4% vs. 54.5% +/- 12.9%), fractional shortening (21.6% +/- 8.1% vs. 24.1% +/- 7.5%), mean velocity of circumferential fiber shortening [(0.8 +/- 0.5) cir/s vs. (0.9 +/- 0.4) cir/s] were significantly increased (P < 0.01), left ventricular end-systolic dimension [(34.0 +/- 8.6) mm vs. (32.2 +/- 9.1) mm] (P < 0.05), left ventricular mass [(65.9 +/- 34.1) g vs. (65.9 +/- 34.1) g], interventricular septal thickness at end-systole [(6.0 +/- 1.0) mm vs (5.5 +/- 1.1) mm] were notably decreased (P < 0.01) after treatment with carvedilol. CONCLUSION: These data indicated that plasma concentration of BNP significantly increased in children with EFE, carvedilol can decrease plasma concentration of BNP, inhibit the remodeling of ventricle, significantly improve the cardiac function in children with EFE. Carvedilol is effective and safe in treatment of children with EFE.