Intramolecular allosteric communication in dopamine D2 receptor revealed by evolutionary amino acid covariation

The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues.Identifying residues that coevolved to maintain or acquire fitness properties is critical for understanding protein structure, function, and evolution (1). Previous studies have shown that covarying residue pairs, those that exhibit correlated amino acid changes in large multiple sequence alignments, tend to form structural contacts (2–7), enhancing predictions of protein 3D structures (8–11). Covariation can also involve distal residues, but the function of these at-a-distance couplings is elusive and has been attributed to background noise, alternative protein conformations, or subunit interactions of protein homooligomers (5, 7, 12). Alternately, distal covarying residue pairs could indicate allosteric couplings (6, 13–18).The possibility of capturing intramolecular allosteric communication by amino acid covariation analysis of protein family sequences has not been extensively explored. Nonproximal thermodynamic coupling between correlated residue pairs was noted in 274 PDZ domains (14), but the relationship to allostery is still debated (19, 20). It may be that distinctive allosteric mechanisms, even among close homologs, limit the extraction of allosteric couplings from sequences (13). Our previous identification of residues important for allosteric signaling within G protein-coupled receptors (GPCRs) using Evolutionary Trace (ET) (21–24) and strong conservation of some of the residues implicated led us to ask whether ET could also uncover couplings among protein sequence positions not in direct contact.ET estimates the relative functional sensitivity of a protein to variations at each residue position using phylogenetic distances to account for the functional divergence among sequence homologs (25, 26). Similar ideas can be applied to pairs of sequence positions to recompute ET as the average importance of the couplings between a residue and its direct structural neighbors (27). To measure the evolutionary coupling information between residue pairs, we present a new algorithm, ET-MIp, that integrates the mutual information metric (MIp) (5) to the ET framework. We used dopamine D2 receptor (D2R), a target of drugs for neurological and psychiatric diseases (28), to test whether ET-MIp could elucidate the allosteric functional communications from amino acid covariation patterns and resolve the evolutionary distance at which the allosteric pathways of D2R homologs are sufficiently conserved to detect residue−residue coupling signatures. D2R is expressed in the central nervous system and responds to dopamine, the major catecholamine neurotransmitter. Canonical D2R signaling is effected by G_i/o class G proteins, which regulate ion channels (29, 30), MAPK kinases (31), phospholipase C (32), and inhibition of adenylyl cyclase (33). D1 class receptors (D1R and D5R) have lower affinities for dopamine (34–36) and activate adenylyl cyclase through G_s class G proteins. To characterize allosteric communication between covarying pairs of residues ranked as important by ET (ET residue pairs), we examined functional coupling for ligand binding affinities and downstream G_i activation induced by agonist-stimulated D2R.     

Effects of dynamic bilevel positive airway pressure support on central sleep apnea in men with heart failure

BACKGROUND: Treatment with continuous positive airway pressure (CPAP) improves cardiac function in chronic heart failure (CHF) patients with central sleep apnea (CSA)-Cheyne-Stokes respiration (CSR) by stabilizing ventilation, but frequently central apneas and hypopneas persist. Our objective was to test the hypothesis that flow-targeted dynamic bilevel positive airway pressure (BPAP) support (BiPAP autoSV; Respironics; Murrysville, PA) effectively suppresses CSR-CSA in CHF patients. METHODS: We studied 14 CHF patients with CSR-CSA (and residual CSA on positive airway pressure therapy) during 3 consecutive nights: (1) diagnostic polysomnography, (2) CPAP (n=10) or BPAP (n=4) titration, and (3) dynamic flow-targeted dynamic BPAP support with an expiratory positive airway pressure (EPAP) set to suppress obstructive respiratory events, and an inspiratory positive airway pressure (IPAP) dynamically ranging between 0 and 15 cm H2O above the EPAP. RESULTS: CPAP or BPAP significantly reduced the apnea-hypopnea index (AHI) [mean+/-SD, 46+/-4 events/h to 22+/-4 events/h; p=0.001] compared to the first night without treatment. Flow-targeted dynamic BPAP support (mean EPAP, 6.5+/-1.7 cm H2O; maximal IPAP, 21.9+/-2.1 cm H2O) further reduced the AHI to 4+/-1/h of sleep compared to the untreated (p<0.001) and CPAP or BPAP night (p=0.002). After the first night of flow-targeted dynamic BPAP support, patients rated on an analog scale (range, 0 to 10) the treatment as comfortable (6.9+/-0.6), and the sleep quality as improved compared to previous nights (7.4+/-0.6). CONCLUSION: Flow-targeted dynamic BPAP support effectively suppresses CSR-CSA in patients with CHF and is well tolerated.     

Clinical efficacy and survival with first-line inhaled iloprost therapy in patients with idiopathic pulmonary arterial hypertension.

AIMS: To describe the long-term clinical efficacy of inhaled iloprost as first-line vasodilator mono-therapy in patients with idiopathic pulmonary arterial hypertension (IPAH). METHODS AND RESULTS: Seventy-six IPAH patients were prospectively identified and treated with inhaled iloprost. Clinical, haemodynamic, and exercise parameters were obtained at baseline, after 3 and 12 months of therapy and yearly thereafter. Four endpoints were prospectively defined as follows: (i) death, (ii) transplantation, (iii) switch to intravenous (i.v.) therapy, or (iv) addition of or switch to other active oral therapy. During follow-up (535+/-61 days), 11 patients died, six were transplanted, 25 were switched to i.v. prostanoids, 16 received additional or other oral therapy, and 12 patients discontinued iloprost inhalation for other reasons. Event-free survival at 3, 12, 24, 36, 48, and 60 months was 81, 53, 29, 20, 17 and 13%, respectively. Among haemodynamic and exercise parameters, mixed venous oxygen saturation (P<0.001), right atrial pressure (P<0.001), and peak oxygen uptake (P=0.002) were associated with event-free survival. CONCLUSION: In this study, only a minority of patients could be stabilized with inhaled iloprost mono-therapy during a follow-up period of up to 5 years. In the presence of multiple treatment options, chronic iloprost inhalation as mono-therapy appears to have a limited role.     

对慢性心力衰竭患者的心肺运动试验采用非线性分析可提高预测价值：摄氧效率斜率

目的:次极量运动过程中摄氧量(VO2)的降低,妨碍了对慢性心力衰竭(CH F)患者依据峰值运动VO2预测生存情况。摄氧效率斜率(OUES)是对运动的通气反应的非线性描绘,甚至在运动早期就可能反映出异常。作者评价了OUES的生理学意义及其对CH F患者预后信息的潜在价值。方法和结果:243例     

ABCA4 mutations causing mislocalization are found frequently in patients with severe retinal dystrophies

ABCA4, also called ABCR, is a retinal-specific member of the ATP-binding cassette (ABC) family that functions in photoreceptor outer segments as a flipase of all-trans retinal. Homozygous and compound heterozygous ABCA4 mutations are associated with various autosomal recessive retinal dystrophies, whereas heterozygous ABCA4 mutations have been associated with dominant susceptibility to age-related macular degeneration in both humans and mice. We analyzed a cohort of 29 arRP families for the mutations in ABCA4 with a commercial microarray, ABCR-400 in addition to direct sequencing and segregation analysis, and identified both mutant alleles in two families (7%): compound heterozygosity for missense (R602W) and nonsense (R408X) alleles and homozygosity for a complex [L541P; A1038V] allele. The missense mutations were analyzed functionally in the photoreceptors of Xenopus laevis tadpoles, which revealed mislocalization of ABCA4 protein. These mutations cause retention of ABCA4 in the photoreceptor inner segment, likely by impairing correct folding, resulting in the total absence of physiologic protein function. Patients with different retinal dystrophies harboring two misfolding alleles exhibit early age-of-onset (AO) (5-12 years) of retinal disease. Our data suggest that a class of ABCA4 mutants may be an important determinant of the AO of disease.