Intriguing role of water in protein-ligand binding studied by neutron crystallography on trypsin complexes.

Hydrogen bonds are key interactions determining protein-ligand binding affinity and therefore fundamental to any biological process. Unfortunately, explicit structural information about hydrogen positions and thus H-bonds in protein-ligand complexes is extremely rare and similarly the important role of water during binding remains poorly understood. Here, we report on neutron structures of trypsin determined at very high resolutions ≤1.5 Å in uncomplexed and inhibited state complemented by X-ray and thermodynamic data and computer simulations. Our structures show the precise geometry of H-bonds between protein and the inhibitors N-amidinopiperidine and benzamidine along with the dynamics of the residual solvation pattern. Prior to binding, the ligand-free binding pocket is occupied by water molecules characterized by a paucity of H-bonds and high mobility resulting in an imperfect hydration of the critical residue Asp189. This phenomenon likely constitutes a key factor fueling ligand binding via water displacement and helps improving our current view on water influencing protein-ligand recognition.

Analysis of the risk factors for early failure after extracardiac Fontan operation.

BACKGROUND: We analyzed risks for severe morbidity in the early period after extracardiac Fontan operation. METHODS: Between November 1995 and May 2011, 140 patients (median age, 3.8 years) underwent extracardiac Fontan operation. We assumed as preoperative risk factors systemic right ventricle (n=51), heterotaxia (n=25), arterial oxygen saturation less than 75% (n=22), and adult age (>16 years, n=20) at time of surgery. Prolonged cardiopulmonary bypass time of longer than 120 minutes (n=30) and use of cardioplegia (n=26) were analyzed as intraoperative risks. RESULTS: Heterotaxia was revealed as a risk factor for postoperative prolonged inotropic support, acute renal failure, prolonged mechanical ventilation, prolonged pleural effusions, and tachyarrhythmias. With the exception of pleural effusions, the same held true for right ventricle morphology. Low preoperative arterial oxygen saturation was found to be associated with an increased risk of prolonged inotropic support, acute renal failure, and prolonged mechanical ventilation. Adult age was identified as a risk factor for acute renal failure. Of the intraoperative factors, prolonged cardiopulmonary bypass time longer than 120 minutes was a risk factor for acute renal failure and prolonged pleural effusions, whereas use of cardioplegia was associated with an increased risk of prolonged inotropic support, prolonged mechanical ventilation, acute renal failure, and tachyarrhythmias. Multivariate analysis demonstrated heterotaxia, right ventricular morphology, and low preoperative arterial oxygen saturation to be independent risk factors for postoperative prolonged inotropic support and prolonged mechanical ventilation. CONCLUSIONS: Patients with heterotaxia, systemic right ventricle, and low preoperative arterial oxygen saturation are still at high risk for early Fontan failure after extracardiac Fontan operation and require special management for optimal outcome.

Improved early postoperative outcome for extracardiac Fontan operation without cardiopulmonary bypass: a single-centre experience.

OBJECTIVES: The use of modified extracardiac Fontan operation (ECFO) for total cavo-pulmonary connection allows cardiopulmonary bypass (CPB) to be avoided and seems to improve early postoperative results. We evaluated our experience with the off-pump technique for ECFO. METHODS: Since 2009, the last 17 consecutive patients of 137 (median age 3.2 years, median weight 14.5 kg) in whom no intracardiac surgery was necessary underwent ECFO without CPB. The non-fenestrated graft was connected end-to-side to the pulmonary artery without bypass; subsequently temporary passive inferior vena cava (IVC)-to-atrial bypass was used for the anastomosis between IVC and graft. The perioperative and postoperative course was compared between consecutive paediatric patients operated on using the CPB vs off-pump technique. RESULTS: There was no mortality in the off-pump group, with a total early mortality of 3.0%. Overall operation time for the Fontan operation using the off-pump technique was significantly reduced (160 vs 200 min, P < 0.001). The median Fontan pressure 24 and 48 h postoperatively was significantly lower in the off-pump group (P = 0.002/0.042). Duration of mechanical ventilation (9 vs 14 h, P = 0.016), pleural effusions (4 vs 8 days, P < 0.001) as well as the median intensive care unit (2 vs 4 days, P = 0.013) and hospital stay (median 10 vs 15 days, P < 0.001) was significantly shorter in patients who underwent the off-pump Fontan operation. The necessity of blood transfusions was significantly reduced with the off-pump in comparison with the on-pump technique (14 of 17 vs 34 of 84 patients, P = 0.003). CONCLUSIONS: The ECFO without CPB is an established low-risk surgical procedure that improves the early postoperative course and significantly reduces the use of blood products and the duration of pleural effusions in selected patients.

Reconstructing the binding site of factor Xa in trypsin reveals ligand-induced structural plasticity.

In order to investigate issues of selectivity and specificity in protein-ligand interactions, we have undertaken the reconstruction of the binding pocket of human factor Xa in the structurally related rat trypsin by site-directed mutagenesis. Three sequential regions (the "99"-, the "175"- and the "190"- loops) were selected as representing the major structural differences between the ligand binding sites of the two enzymes. Wild-type rat trypsin and variants X99rT and X(99/175/190)rT were expressed in yeast, and analysed for their interaction with factor Xa and trypsin inhibitors. For most of the inhibitors studied, progressive loop replacement at the trypsin surface resulted in inhibitory profiles akin to factor Xa. Crystals of the variants were obtained in the presence of benzamidine (3), and could be soaked with the highly specific factor Xa inhibitor (1). Binding of the latter to X99rT results in a series of structural adaptations to the ligand, including the establishment of an "aromatic box" characteristic of factor Xa. In X(99/175/190)rT, introduction of the 175-loop results in a surprising re-orientation of the "intermediate helix", otherwise common to trypsin and factor Xa. The re-orientation is accompanied by an isomerisation of the Cys168-Cys182 disulphide bond, and burial of the critical Phe174 side-chain. In the presence of (1), a major re-organisation of the binding site takes place to yield a geometry identical to that of factor Xa. In all, binding of (1) to trypsin and its variants results in significant structural rearrangements, inducing a binding surface strongly reminiscent of factor Xa, against which the inhibitor was optimised. The structural data reveal a plasticity of the intermediate helix, which has been implicated in the functional cofactor dependency of many trypsin-like serine proteinases. This approach of grafting loops onto scaffolds of known related structures may serve to bridge the gap between structural genomics and drug design.