Sequence - dynamics - function relationships in protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) are crucial regulators of cellular signaling. Their activity is regulated by the motion of a conserved loop, the WPD-loop, from a catalytically inactive open to a catalytically active closed conformation. WPD-loop motion optimally positions a catalytically critical residue into the active site, and is directly linked to the turnover number of these enzymes. Crystal structures of chimeric PTPs constructed by grafting parts of the WPD-loop sequence of PTP1B onto the scaffold of YopH showed WPD-loops in a wide-open conformation never previously observed in either parent enzyme. This wide-open conformation has, however, been observed upon binding of small molecule inhibitors to other PTPs, suggesting the potential of targeting it for drug discovery efforts. Here, we have performed simulations of both enzymes and show that there are negligible energetic differences in the chemical step of catalysis, but significant differences in the dynamical properties of the WPD-loop. Detailed interaction network analysis provides insight into the molecular basis for this population shift to a wide-open conformation. Taken together, our study provides insight into the links between loop dynamics and chemistry in these YopH variants specifically, and how WPD-loop dynamic can be engineered through modification of the internal protein interaction network.

Insights into the importance of WPD-loop sequence for activity and structure in protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) possess a conserved mobile catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. We have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics. The chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, and show differences in the pH dependency of catalysis, and changes in the effect of Mg2+. The chimeric proteins' WPD-loops differ significantly in their relative stability and rigidity. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.

GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases.

GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the Gαi subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2PO4- and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) "undruggable" GTPases like Ras.

Influence of Frozen Residues on the Exploration of the PES of Enzyme Reaction Mechanisms.

In this work, we studied one of the very widely used approximations in the prediction of an enzyme reaction mechanism with computational methods, that is, fixing residues outside a given radius surrounding the active site. This avoids the unfolding of truncated models during MD calculations, avoids the expansion of the active site in cluster model calculations (albeit here only specific atoms are frozen), and prevents drifting between local minima when adiabatic mapping with large QM/MM models is used. To test this, we have used the first step of the reaction catalyzed by HIV-1 protease, as the detrimental effects of this approximation are expected to be large here. We calculated the PES with shells of frozen residues of different radii. Models with free regions under a 6.00 Å radius showed signs of being overconstrained. The QM/MM energy barrier for the remaining models was only slightly sensitive to this approximation (average of 0.8 kcal·mol-1, maximum of 1.6 kcal·mol-1). The influence over the energy of reaction was almost negligible. This widely used approximation seems safe and robust. The resulting error is on average below 1.6 kcal·mol-1, which is small when compared with others deriving from, for example, the choice of the density functional or semiempirical MO/SCC-DFTB method, the basis set used, or even the lack of sampling or incomplete sampling.

Professional Competencies of Cuban Specialists in Intensive Care and Emergency Medicine.

INTRODUCTION The quality of medical training and practice reflects the competency level of the professionals involved. The intensive care and emergency medicine specialty in Cuba has not defined its competencies. OBJECTIVE Identify the competencies required for specialty practice in intensive care and emergency medicine. METHODS The study was conducted from January 2014 to December 2015, using qualitative techniques; 48 professionals participated. We undertook functional occupational analysis, based on functions defined in a previous study. Three expert groups were utilized: the first used various group techniques; the second, the Delphi method; and the third, the Delphi method and a Likert questionnaire. RESULTS A total of 73 specific competencies were defined, grouped in 11 units: 44 in the patient care function, 16 in management, 7 in teaching and 6 in research. A competency map is provided. CONCLUSIONS The intensive care and emergency medicine specialty competencies identified will help improve professional standards, ensure health workforce quality, improve patient care and academic performance, and enable objective evaluation of specialists' competence and performance. KEYWORDS Clinical competency, competency-based education, professional education, intensive care, emergency medicine, urgent care, continuing medical education, curriculum, medical residency, Cuba.

The Catalytic Mechanism of the Marine-Derived Macrocyclase PatGmac.

Cyclic peptides are a class of compounds with high therapeutic potential, possessing bioactivities including antitumor and antiviral (including anti-HIV). Despite their desirability, efficient design and production of these compounds has not been achieved to date. The catalytic mechanism of patellamide macrocyclization by the PatG macrocyclase domain has been computationally investigated by using quantum mechanics/molecular mechanics methodology, specifically ONIOM(M06/6-311++G(2d,2p):ff94//B3LYP/6-31G(d):ff94). The mechanism proposed herein begins with a proton transfer from Ser783 to His 618 and from the latter to Asp548. Nucleophilic attack of Ser783 on the substrate leads to the formation of an acyl-enzyme covalent complex. The leaving group Ala-Tyr-Asp-Gly (AYDG) of the substrate is protonated by the substrate's N terminus, leading to the breakage of the P1-P1' bond. Finally, the substrate's N terminus attacks the P1 residue, decomposing the acyl-enzyme complex forming the macrocycle. The formation and decomposition of the acyl-enzyme complex have the highest activation free energies (21.1 kcal mol(-1) and 19.8 kcal mol(-1) respectively), typical of serine proteases. Understanding the mechanism behind the macrocyclization of patellamides will be important to the application of the enzymes in the pharmaceutical and biotechnological industries.