Experimental Analysis of Functional Variation within Protein Families: Receiver Domain Autodephosphorylation Kinetics.

UNLABELLED: Plants and microorganisms use two-component signal transduction systems (TCSs) to mediate responses to environmental stimuli. TCSs mediate responses through phosphotransfer from a conserved histidine on a sensor kinase to a conserved aspartate on the receiver domain of a response regulator. Typically, signal termination occurs through dephosphorylation of the receiver domain, which can catalyze its own dephosphorylation. Despite strong structural conservation between receiver domains, reported autodephosphorylation rate constants (kdephos) span a millionfold range. Variable receiver domain active-site residues D + 2 and T + 2 (two amino acids C terminal to conserved phosphorylation site and Thr/Ser, respectively) influence kdephos values, but the extent and mechanism of influence are unclear. We used sequence analysis of a large database of naturally occurring receiver domains to design mutant receiver domains for experimental analysis of autodephosphorylation kinetics. When combined with previous analyses, kdephos values were obtained for CheY variants that contained D + 2/T + 2 pairs found in 54% of receiver domain sequences. Tested pairs of amino acids at D + 2/T + 2 generally had similar effects on kdephos in CheY, PhoBN, or Spo0F. Acid or amide residues at D + 2/T + 2 enhanced kdephos CheY variants altered at D + 2/T + 2 exhibited rate constants for autophosphorylation with phosphoramidates and autodephosphorylation that were inversely correlated, suggesting that D + 2/T + 2 residues interact with aspects of the ground or transition states that differ between the two reactions. kdephos of CheY variants altered at D + 2/T + 2 correlated significantly with kdephos of wild-type receiver domains containing the same D + 2/T + 2 pair. Additionally, particular D + 2/T + 2 pairs were enriched in different response regulator subfamilies, suggesting functional significance. IMPORTANCE: One protein family, defined by a conserved domain, can include hundreds of thousands of known members. Characterizing conserved residues within a conserved domain can identify functions shared by all family members. However, a general strategy to assess features that differ between members of a family is lacking. Fully exploring the impact of just two variable positions within a conserved domain could require assessment of 400 (i.e., 20 × 20) variants. Instead, we created and analyzed a nonredundant database of receiver domain sequences. Five percent of D + 2/T + 2 pairs were sufficient to represent 50% of receiver domain sequences. Using protein sequence analysis to prioritize mutant choice made it experimentally feasible to extensively probe the influence of positions D + 2 and T + 2 on receiver domain autodephosphorylation kinetics.

Superior outcomes and lower outpatient costs with scale-up of antiretroviral therapy at the GHESKIO clinic in Port-au-Prince, Haiti.

BACKGROUND: Treatment protocols and prices of antiretroviral therapy (ART) have changed over time. Yet, limited data exist to evaluate the impact of these changes on patient outcomes and treatment costs in resource-poor settings. METHODS: We compared patient-level data on outcomes, utilization, and cost for the first 2 years of ART for a cohort of adult patients initiating ART in 2003-2004 and a cohort initiating ART in 2006-2008 at the Haitian Group for the Study of Kaposi's Sarcoma and Opportunistic Infections clinic (GHESKIO) in Port-au-Prince, Haiti. Costs were measured from the health center perspective. Multivariate analyses were conducted to account for the potential impact of differences in disease severity at baseline. RESULTS: With the exclusion of patients who transferred care, 92% (167/181) of patients in the 2006-2008 cohort and 75% (150/200) in the 2003-2004 cohort were alive and in care at the end of the study period. The mean cost per patient for the 2-year study period was US$723 for the 2006-2008 cohort vs. US$1191 for the 2003-2004 cohort, a cost difference of US$468 (P < 0.0001). The mean cost per patient alive and in care at the end of the 2-year study period was US$744 for the 2006-2008 cohort vs. US$1489 for the 2003-2004 cohort (P < 0.0001). CONCLUSIONS: HIV treatment outcomes in Haiti have improved over time while treatment costs declined by over 50% per patient alive and in care at the end of the 2-year study period. The major drivers in the reduction of treatment costs were the lower price of ART, lower costs for laboratory testing, and lower overhead costs.