Installing hydrolytic activity into a completely <i>de novo </i>protein framework

The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine–histidine–glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis

Specificity quantification of biomolecular recognition and its implication for drug discovery

Highly efficient and specific biomolecular recognition requires both affinity and specificity. Previous quantitative descriptions of biomolecular recognition were mostly driven by improving the affinity prediction, but lack of quantification of specificity. We developed a novel method SPA (SPecificity and Affinity) based on our funneled energy landscape theory. The strategy is to simultaneously optimize the quantified specificity of the “native” protein-ligand complex discriminating against “non-native” binding modes and the affinity prediction. The benchmark testing of SPA shows the best performance against 16 other popular scoring functions in industry and academia on both prediction of binding affinity and “native” binding pose. For the target COX-2 of nonsteroidal anti-inflammatory drugs, SPA successfully discriminates the drugs from the diversity set, and the selective drugs from non-selective drugs. The remarkable performance demonstrates that SPA has significant potential applications in identifying lead compounds for drug discovery

How Protein Stability and New Functions Trade Off

Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (ΔΔG), thus suggesting a tradeoff between the acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (ΔΔG>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied ΔΔG computations by FoldX to analyze the effects of 548 mutations that arose from the directed evolution of 22 different enzymes. The stability effects, location, and type of function-altering mutations were compared to ΔΔG changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average ΔΔG = +0.9 kcal/mol), and are almost as destabilizing as the “average” mutation in these enzymes (+1.3 kcal/mol). Although their stability effects are not as dramatic as in key catalytic residues, mutations that modify the substrate binding pockets, and thus mediate new enzymatic specificities, place a larger stability burden than surface mutations that underline neutral, non-adaptive evolutionary changes. How are the destabilizing effects of functional mutations balanced to enable adaptation? Our analysis also indicated that many mutations that appear in directed evolution variants with no obvious role in the new function exert stabilizing effects that may compensate for the destabilizing effects of the crucial function-altering mutations. Thus, the evolution of new enzymatic activities, both in nature and in the laboratory, is dependent on the compensatory, stabilizing effect of apparently “silent” mutations in regions of the protein that are irrelevant to its function

A Measure of the Promiscuity of Proteins and Characteristics of Residues in the Vicinity of the Catalytic Site That Regulate Promiscuity

Promiscuity, the basis for the evolution of new functions through ‘tinkering’ of residues in the vicinity of the catalytic site, is yet to be quantitatively defined. We present a computational method Promiscuity Indices Estimator (PROMISE) - based on signatures derived from the spatial and electrostatic properties of the catalytic residues, to estimate the promiscuity (PromIndex) of proteins with known active site residues and 3D structure. PromIndex reflects the number of different active site signatures that have congruent matches in close proximity of its native catalytic site, the quality of the matches and difference in the enzymatic activity. Promiscuity in proteins is observed to follow a lognormal distribution (μ = 0.28, σ = 1.1 reduced chi-square = 3.0E-5). The PROMISE predicted promiscuous functions in any protein can serve as the starting point for directed evolution experiments. PROMISE ranks carboxypeptidase A and ribonuclease A amongst the more promiscuous proteins. We have also investigated the properties of the residues in the vicinity of the catalytic site that regulates its promiscuity. Linear regression establishes a weak correlation (R2∼0.1) between certain properties of the residues (charge, polar, etc) in the neighborhood of the catalytic residues and PromIndex. A stronger relationship states that most proteins with high promiscuity have high percentages of charged and polar residues within a radius of 3 Å of the catalytic site, which is validated using one-tailed hypothesis tests (P-values∼0.05). Since it is known that these characteristics are key factors in catalysis, their relationship with the promiscuity index cross validates the methodology of PROMISE

Tradeoff Between Stability and Multispecificity in the Design of Promiscuous Proteins

Natural proteins often partake in several highly specific protein-protein interactions. They are thus subject to multiple opposing forces during evolutionary selection. To be functional, such multispecific proteins need to be stable in complex with each interaction partner, and, at the same time, to maintain affinity toward all partners. How is this multispecificity acquired through natural evolution? To answer this compelling question, we study a prototypical multispecific protein, calmodulin (CaM), which has evolved to interact with hundreds of target proteins. Starting from high-resolution structures of sixteen CaM-target complexes, we employ state-of-the-art computational methods to predict a hundred CaM sequences best suited for interaction with each individual CaM target. Then, we design CaM sequences most compatible with each possible combination of two, three, and all sixteen targets simultaneously, producing almost 70,000 low energy CaM sequences. By comparing these sequences and their energies, we gain insight into how nature has managed to find the compromise between the need for favorable interaction energies and the need for multispecificity. We observe that designing for more partners simultaneously yields CaM sequences that better match natural sequence profiles, thus emphasizing the importance of such strategies in nature. Furthermore, we show that the CaM binding interface can be nicely partitioned into positions that are critical for the affinity of all CaM-target complexes and those that are molded to provide interaction specificity. We reveal several basic categories of sequence-level tradeoffs that enable the compromise necessary for the promiscuity of this protein. We also thoroughly quantify the tradeoff between interaction energetics and multispecificity and find that facilitating seemingly competing interactions requires only a small deviation from optimal energies. We conclude that multispecific proteins have been subjected to a rigorous optimization process that has fine-tuned their sequences for interactions with a precise set of targets, thus conferring their multiple cellular functions

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

Artificial endonucleases consisting of a Fokl cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off- target sites. Here we describe a structure- based approach to reducing off- target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology

Spontaneous Breathing in Early Acute Respiratory Distress Syndrome: Insights From the Large Observational Study to UNderstand the Global Impact of Severe Acute Respiratory FailurE Study

OBJECTIVES: To describe the characteristics and outcomes of patients with acute respiratory distress syndrome with or without spontaneous breathing and to investigate whether the effects of spontaneous breathing on outcome depend on acute respiratory distress syndrome severity. DESIGN: Planned secondary analysis of a prospective, observational, multicentre cohort study. SETTING: International sample of 459 ICUs from 50 countries. PATIENTS: Patients with acute respiratory distress syndrome and at least 2 days of invasive mechanical ventilation and available data for the mode of mechanical ventilation and respiratory rate for the 2 first days. INTERVENTIONS: Analysis of patients with and without spontaneous breathing, defined by the mode of mechanical ventilation and by actual respiratory rate compared with set respiratory rate during the first 48 hours of mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Spontaneous breathing was present in 67% of patients with mild acute respiratory distress syndrome, 58% of patients with moderate acute respiratory distress syndrome, and 46% of patients with severe acute respiratory distress syndrome. Patients with spontaneous breathing were older and had lower acute respiratory distress syndrome severity, Sequential Organ Failure Assessment scores, ICU and hospital mortality, and were less likely to be diagnosed with acute respiratory distress syndrome by clinicians. In adjusted analysis, spontaneous breathing during the first 2 days was not associated with an effect on ICU or hospital mortality (33% vs 37%; odds ratio, 1.18 [0.92-1.51]; p = 0.19 and 37% vs 41%; odds ratio, 1.18 [0.93-1.50]; p = 0.196, respectively ). Spontaneous breathing was associated with increased ventilator-free days (13 [0-22] vs 8 [0-20]; p = 0.014) and shorter duration of ICU stay (11 [6-20] vs 12 [7-22]; p = 0.04). CONCLUSIONS: Spontaneous breathing is common in patients with acute respiratory distress syndrome during the first 48 hours of mechanical ventilation. Spontaneous breathing is not associated with worse outcomes and may hasten liberation from the ventilator and from ICU. Although these results support the use of spontaneous breathing in patients with acute respiratory distress syndrome independent of acute respiratory distress syndrome severity, the use of controlled ventilation indicates a bias toward use in patients with higher disease severity. In addition, because the lack of reliable data on inspiratory effort in our study, prospective studies incorporating the magnitude of inspiratory effort and adjusting for all potential severity confounders are required

Identifying associations between diabetes and acute respiratory distress syndrome in patients with acute hypoxemic respiratory failure: an analysis of the LUNG SAFE database

Background: Diabetes mellitus is a common co-existing disease in the critically ill. Diabetes mellitus may reduce the risk of acute respiratory distress syndrome (ARDS), but data from previous studies are conflicting. The objective of this study was to evaluate associations between pre-existing diabetes mellitus and ARDS in critically ill patients with acute hypoxemic respiratory failure (AHRF). Methods: An ancillary analysis of a global, multi-centre prospective observational study (LUNG SAFE) was undertaken. LUNG SAFE evaluated all patients admitted to an intensive care unit (ICU) over a 4-week period, that required mechanical ventilation and met AHRF criteria. Patients who had their AHRF fully explained by cardiac failure were excluded. Important clinical characteristics were included in a stepwise selection approach (forward and backward selection combined with a significance level of 0.05) to identify a set of independent variables associated with having ARDS at any time, developing ARDS (defined as ARDS occurring after day 2 from meeting AHRF criteria) and with hospital mortality. Furthermore, propensity score analysis was undertaken to account for the differences in baseline characteristics between patients with and without diabetes mellitus, and the association between diabetes mellitus and outcomes of interest was assessed on matched samples. Results: Of the 4107 patients with AHRF included in this study, 3022 (73.6%) patients fulfilled ARDS criteria at admission or developed ARDS during their ICU stay. Diabetes mellitus was a pre-existing co-morbidity in 913 patients (22.2% of patients with AHRF). In multivariable analysis, there was no association between diabetes mellitus and having ARDS (OR 0.93 (0.78-1.11); p = 0.39), developing ARDS late (OR 0.79 (0.54-1.15); p = 0.22), or hospital mortality in patients with ARDS (1.15 (0.93-1.42); p = 0.19). In a matched sample of patients, there was no association between diabetes mellitus and outcomes of interest. Conclusions: In a large, global observational study of patients with AHRF, no association was found between diabetes mellitus and having ARDS, developing ARDS, or outcomes from ARDS. Trial registration: NCT02010073. Registered on 12 December 2013

Epidemiology and patterns of tracheostomy practice in patients with acute respiratory distress syndrome in ICUs across 50 countries

Background: To better understand the epidemiology and patterns of tracheostomy practice for patients with acute respiratory distress syndrome (ARDS), we investigated the current usage of tracheostomy in patients with ARDS recruited into the Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG-SAFE) study. Methods: This is a secondary analysis of LUNG-SAFE, an international, multicenter, prospective cohort study of patients receiving invasive or noninvasive ventilation in 50 countries spanning 5 continents. The study was carried out over 4 weeks consecutively in the winter of 2014, and 459 ICUs participated. We evaluated the clinical characteristics, management and outcomes of patients that received tracheostomy, in the cohort of patients that developed ARDS on day 1-2 of acute hypoxemic respiratory failure, and in a subsequent propensity-matched cohort. Results: Of the 2377 patients with ARDS that fulfilled the inclusion criteria, 309 (13.0%) underwent tracheostomy during their ICU stay. Patients from high-income European countries (n = 198/1263) more frequently underwent tracheostomy compared to patients from non-European high-income countries (n = 63/649) or patients from middle-income countries (n = 48/465). Only 86/309 (27.8%) underwent tracheostomy on or before day 7, while the median timing of tracheostomy was 14 (Q1-Q3, 7-21) days after onset of ARDS. In the subsample matched by propensity score, ICU and hospital stay were longer in patients with tracheostomy. While patients with tracheostomy had the highest survival probability, there was no difference in 60-day or 90-day mortality in either the patient subgroup that survived for at least 5 days in ICU, or in the propensity-matched subsample. Conclusions: Most patients that receive tracheostomy do so after the first week of critical illness. Tracheostomy may prolong patient survival but does not reduce 60-day or 90-day mortality. Trial registration: ClinicalTrials.gov, NCT02010073. Registered on 12 December 2013