From Steroid and Drug Metabolism to Glycobiology, Using Sulfotransferase Structures to Understand and Tailor Function.

Sulfotransferases are ubiquitous enzymes that transfer a sulfo group from the universal cofactor donor 3'-phosphoadenosine 5'-phosphosulfate to a broad range of acceptor substrates. In humans, the cytosolic sulfotransferases are involved in the sulfation of endogenous compounds such as steroids, neurotransmitters, hormones, and bile acids as well as xenobiotics including drugs, toxins, and environmental chemicals. The Golgi associated membrane-bound sulfotransferases are involved in post-translational modification of macromolecules from glycosaminoglycans to proteins. The sulfation of small molecules can have profound biologic effects on the functionality of the acceptor, including activation, deactivation, or enhanced metabolism and elimination. Sulfation of macromolecules has been shown to regulate a number of physiologic and pathophysiological pathways by enhancing binding affinity to regulatory proteins or binding partners. Over the last 25 years, crystal structures of these enzymes have provided a wealth of information on the mechanisms of this process and the specificity of these enzymes. This review will focus on the general commonalities of the sulfotransferases, from enzyme structure to catalytic mechanism as well as providing examples into how structural information is being used to either design drugs that inhibit sulfotransferases or to modify the enzymes to improve drug synthesis. SIGNIFICANCE STATEMENT: This manuscript honors Dr. Masahiko Negishi's contribution to the understanding of sulfotransferase mechanism, specificity, and roles in biology by analyzing the crystal structures that have been solved over the last 25 years.

Preferential DNA Polymerase ß Reverse Reaction with Imidodiphosphate.

DNA replication and repair reactions involve the addition of a deoxynucleoside monophosphate onto a growing DNA strand with the loss of pyrophosphate. This chemical reaction is also reversible; the addition of pyrophosphate generates a deoxynucleoside triphosphate, thereby shortening the DNA by one nucleotide. The forward DNA synthesis and reverse pyrophosphorolysis reactions strictly require the presence of divalent metals, usually magnesium, at the reactive center as cofactors. The overall equilibrium enzymatic reaction strongly favors DNA synthesis over pyrophosphorolysis with natural substrates. The DNA polymerase ß chemical reaction has been structurally and kinetically characterized, employing natural and chemically modified substrates. Substituting an imido-moiety (NH) for the bridging oxygen between Pß and Pγ of dGTP dramatically decreased the overall enzymatic activity and resulted in a chemical equilibrium that strongly favors the reverse reaction (i.e., K ≪ 1). Using QM/MM calculations in conjunction with the utilization of parameters such as quantum mechanically derived atomic charges, we have examined the chemical foundation for the altered equilibrium with this central biological reaction. The calculations indicate that the rapid reverse reaction is likely due, in part, to the increased nucleophilicity of the reactive oxygen on the tautomeric form of imidodiphosphate.

Ligand induced dissociation of the AR homodimer precedes AR monomer translocation to the nucleus.

The androgen receptor (AR) regulates male sexual development. We have now investigated AR homodimerization, hormone-dependent monomerization and nuclear translocation in PC-3 and COS-1 cells, by utilizing mutations associated with the androgen insensitivity syndrome: Pro767Ala, Phe765Leu, Met743Val and Trp742Arg. AR wild type (WT) was expressed as a homodimer in the cytoplasm, while none of these mutants formed homodimers. Unlike AR WT which responded to 1 nM dihydrotestosterone (DHT) to dissociate and translocate into the nucleus, AR Pro767Ala and Phe765Leu mutants remain as the monomer in the cytoplasm. In the crystal structure of the AR LBD homodimer, Pro767 and Phe765 reside closely on a loop that constitutes the dimer interface; their sidechains interact with the Pro767 of the other monomer and with the DHT molecule in the ligand-binding pocket. These observations place Phe765 at a position to facilitate DHT binding to Pro767 and lead to dissociation of the AR homodimer in the cytoplasm. This Pro-Phe Met relay may constitute a structural switch that mediates androgen signaling and is conserved in other steroid hormone receptors.

Dysfunctional endogenous FIX impairs prophylaxis in a mouse hemophilia B model.

Factor IX (FIX) binds to collagen IV (Col4) in the subendothelial basement membrane. In hemophilia B, this FIX-Col4 interaction reduces the plasma recovery of infused FIX and plays a role in hemostasis. Studies examining the recovery of infused BeneFix (FIXWT) in null (cross-reactive material negative, CRM-) hemophilia B mice suggest the concentration of Col4 readily available for binding FIX is ∼405 nM with a 95% confidence interval of 374 to 436 nM. Thus, the vascular cache of FIX bound to Col4 is several-fold the FIX level measured in plasma. In a mouse model of prophylactic therapy (testing hemostasis by saphenous vein bleeding 7 days after infusion of 150 IU/kg FIX), FIXWT and the increased half-life FIXs Alprolix (FIXFC) and Idelvion (FIXAlb) produce comparable hemostatic results in CRM- mice. In bleeding CRM- hemophilia B mice, the times to first clot at a saphenous vein injury site after the infusions of the FIX agents are significantly different, at FIXWT < FIXFC < FIXAlb Dysfunctional forms of FIX, however, circulate in the majority of patients with hemophilia B (CRM+). In the mouse prophylactic therapy model, none of the FIX products improves hemostasis in CRM+ mice expressing a dysfunctional FIX, FIXR333Q, that nevertheless competes with infused FIX for Col4 binding and potentially other processes involving FIX. The results in this mouse model of CRM+ hemophilia B demonstrate that the endogenous expression of a dysfunctional FIX can deleteriously affect the hemostatic response to prophylactic therapy.

Evaluating the impact of national education in pediatric palliative care: the Quality of Care Collaborative Australia.

PURPOSE: The Quality of Care Collaborative Australia (QuoCCA) provided pediatric palliative care education across Australia with the aim of improving the quality of services. The education was delivered through a collaboration of six tertiary pediatric palliative care services, through funding for Nurse Educators, Medical Fellows, a National Allied Health Educator, and national project staff. METHODS: Pre- and post-education surveys were completed by participants immediately following the education, and confidence and knowledge were measured along nine domains related to the care of the child and family, including managing a new referral, symptom management, medications, preparing the family, and using local agencies. RESULTS: Education was provided to over 5,500 health and human service professionals in 337 education sessions across Australia between May 2015 and June 2017. Paired pre- and post-surveys were completed by 969 participants and showed a significant improvement in all the domains measured. Those with no experience in caring for children receiving palliative care showed greater improvement following QuoCCA education compared to those with experience, although the latter had higher scores both before and after education. Similarly, those with no previous education showed greater improvement, but those with previous education showed higher scores overall. Participants in full-day and half-day sessions showed greater improvement than those in short day sessions. Thus, the dosage of education in the length of the sessions and prior attendance impacted knowledge and confidence. Topics requested by the participants were analyzed. Educator learnings were that education was more effective when tailored to the needs of the audience, was interactive, and included story-telling, case studies, and parent experiences. CONCLUSION: These results encouraged the continuation of the provision of education to novice and experienced professionals who care for children with a life-limiting condition, leading to higher levels of confidence and knowledge. The learnings from this evaluation will be transferred into the second round of funding for the national QuoCCA education project. The next stage will focus on developing simulation and interactive training, accessible training modules, and videos on a national website.

Warfarin and vitamin K epoxide reductase: a molecular accounting for observed inhibition.

Vitamin K epoxide reductase (VKOR), an endoplasmic reticulum membrane protein, is the key enzyme for vitamin K-dependent carboxylation, a posttranslational modification that is essential for the biological functions of coagulation factors. VKOR is the target of the most widely prescribed oral anticoagulant, warfarin. However, the topological structure of VKOR and the mechanism of warfarin's inhibition of VKOR remain elusive. Additionally, it is not clear why warfarin-resistant VKOR mutations identified in patients significantly decrease warfarin's binding affinity, but have only a minor effect on vitamin K binding. Here, we used immunofluorescence confocal imaging of VKOR in live mammalian cells and PEGylation of VKOR's endogenous cytoplasmic-accessible cysteines in intact microsomes to probe the membrane topology of human VKOR. Our results show that the disputed loop sequence between the first and second transmembrane (TM) domain of VKOR is located in the cytoplasm, supporting a 3-TM topological structure of human VKOR. Using molecular dynamics (MD) simulations, a T-shaped stacking interaction between warfarin and tyrosine residue 139, within the proposed TY139A warfarin-binding motif, was observed. Furthermore, a reversible dynamic warfarin-binding pocket opening and conformational changes were observed when warfarin binds to VKOR. Several residues (Y25, A26, and Y139) were found essential for warfarin binding to VKOR by MD simulations, and these were confirmed by the functional study of VKOR and its mutants in their native milieu using a cell-based assay. Our findings provide new insights into the dynamics of the binding of warfarin to VKOR, as well as into warfarin's mechanism of anticoagulation.

Preparing Pediatric Healthcare Professionals for End-of-Life Care Discussions: An Exploratory Study.

BACKGROUND: Preparedness to initiate end-of-life (EoL) discussions is a confronting and daunting task for all healthcare professionals. We conducted a group interview to explore healthcare professionals' experiences of preparing for EoL discussions with the patient and their family in a pediatric context. AIM: To identify what pediatric healthcare professionals consider important when preparing for an EoL discussion. METHODS: A qualitative design using a group interview. Two open-ended questions were asked: (1) How could preparedness to initiate EoL care discussions between healthcare professionals and the patient and family be enhanced? (2) What education resources/strategies could be developed to support preparation for EoL care discussions? SETTING/SUBJECTS: Healthcare professionals, including medical, nursing, and allied health professionals working in pediatric palliative care settings across Queensland, Australia. These settings included major tertiary hospitals, general practice, community, and nongovernment organizations. A convenience sample of 36 healthcare professionals consented to participate in the study. RESULTS: An analysis of the data identified seven themes that had relevance for preparing for an EoL discussion: communication, healthcare professional perspectives, interdisciplinary team role, patient and family perspectives, practical issues, addressing mistakes, and healthcare professional education. CONCLUSIONS: Pediatric healthcare professionals confirmed that gaps exist in preparing for an EoL discussion. The findings support a need for further research in two areas. First, a systematic review of interdisciplinary resources that are available to support healthcare professionals in preparing for EoL discussions is recommended. Second, evidence-based interdisciplinary interventions to support pediatric EoL discussions need to be developed and evaluated.

Revealing the role of the product metal in DNA polymerase ß catalysis.

DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase ß, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction.

Hiding in Plain Sight: The Bimetallic Magnesium Covalent Bond in Enzyme Active Sites.

The transfer of phosphate groups is an essential function of many intracellular biological enzymes. The transfer is in many cases facilitated by a protein scaffold involving two closely spaced magnesium "ions". It has long been a mystery how these "ions" can retain their closely spaced positions throughout enzymatic phosphate transfer: Coulomb's law would dictate large repulsive forces between these ions at the observed distances. Here we show, however, that the electron density can be borrowed from nearby electron-rich oxygens to populate a bonding molecular orbital that is largely localized between the magnesium "ions". The result is that the Mg-Mg core of these phosphate transfer enzymes is surprisingly similar to a metastable [Mg2]2+ ion in the gas phase, an ion that has been identified experimentally and studied with high-level quantum-mechanical calculations. This similarity is confirmed by comparative computations of the electron densities of [Mg2]2+ in the gas phase and the Mg-Mg core in the structures derived from QM/MM studies of high-resolution X-ray crystal structures. That there is a level of covalent bonding between the two Mg "ions" at the core of these enzymes is a novel concept that enables an improved vision of how these enzymes function at the molecular level. The concept is broader than magnesium-other biologically relevant metals (e.g., Mn and Zn) can also form similar stabilizing covalent Me-Me bonds in both organometallic and inorganic crystals.

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse.

DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is "balanced," as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3' of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase ß active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability.

Do the crystallographic forms of prethrombin-2 revert to a single form in solution?

It has been earlier established (Pozzi et al. Biochemistry 50 (2011) 10195-10202) that prethrombin-2 crystallizes into two similar but distinct forms: a collapsed form and an alternative form. We employed long molecular dynamics (MD) simulations for these two forms to obtain solvent-equilibrated forms. We find that, at 200ns, the simulated solution collapsed form is quite similar to the X-ray crystal collapsed form, while the simulated solution alternative form deviates from the X-ray crystal alternative form as well as from the solution collapsed form. A detailed structural analysis suggests that the fluctuation of the 140s-loop, in cross-talk with the 220s-loop, may alter the conformation of the W215-E217 segment near the nascent thrombin active site. A rationale is provided for the manner in which interactions of prethrombin-2 with FVa may affect the equilibrium between the two forms of prethrombin-2.

A model for the unique role of factor Va A2 domain extension in the human ternary thrombin-generating complex.

An all-atom human ternary model for the prothrombinase-prothrombin complex, including metal ions and post-translationally modified residues, was constructed from existing X-ray crystal structures. The factor Xa-prothrombin interface was taken from an existing ternary model, which locates the active site of factor Xa in the vicinity of prothrombin cleavage positions. The three sulfotyrosine residues at the C-terminal sequence of factor Va A2 domain are accommodated by modelling rational interactions with positively charged patches on the surface of prothrombin. The entire model is then solvent-equilibrated with molecular dynamics. This ternary model for the thrombin-generating complex provides an estimate as to the role of the C-terminus of the factor Va A2 domain: to establish an interface between FXa and prothrombin and to stabilize the orientation of this interface.

Applications of quantum mechanical/molecular mechanical methods to the chemical insertion step of DNA and RNA polymerization.

We review theoretical attempts to model the chemical insertion reactions of nucleoside triphosphates catalyzed by the nucleic acid polymerases using combined quantum mechanical/molecular mechanical methodology. Due to an existing excellent database of high-resolution X-ray crystal structures, the DNA polymerase ß system serves as a useful template for discussion and comparison. The convergence of structures of high-quality complexes and continued developments of theoretical techniques suggest a bright future for understanding the global features of nucleic acid polymerization.

Analysis on long-range residue-residue communication using molecular dynamics.

We investigated the possibility of inter-residue communication of side chains in barstar, an 89 residue protein, using mutual information theory. The normalized mutual information (NMI) of the dihedral angles of the side chains was obtained from all-atom molecular dynamics simulations. The accumulated NMI from an explicit solvent equilibrated trajectory (600 ns) with free backbone exhibits a parabola-shaped distribution over the inter-residue distances (0-36 Å): smaller at the end regimes but larger in the middle regime. This analysis, plus several other measures, does not find unusual long-range communication for free backbone in explicit solvent simulations.

Development of a state-wide pediatric palliative care service in Australia: referral and outcomes over two years.

BACKGROUND: Pediatric palliative care is an evolving specialty that aims to improve the lives of children with a life-limiting condition. As an emerging specialty, there is much to be learned about service provision and the expected outcomes that can be achieved. Additionally, quantification of the needs for pediatric palliative care is complicated by the uncertainty of defining the population that requires care. OBJECTIVES: Our aim was to define the characteristics of the population cared for by a newly formed state-wide service in Queensland, Australia, and describe the development of the service over a 24-month period. DESIGN: Data on all referrals and outcomes were collected. Descriptive statistics were used to describe patterns including the variation in outcomes between children with oncology and non-oncology diagnoses. Other factors influencing the development of the service including involved health professionals and the model of the Australian health care system are also described. RESULTS: Over a 24-month period, 150 patients were referred of whom 117 subsequently died. There was a wide range of diseases and ages, and significantly, 58% of children were from regional or rural locations where there are can be limited access to specialist pediatric services. The average length of service was 83 days. A variety of factors were identified as being important for providing optimal care including ensuring equity in access, timing of referral, and continuity of care. CONCLUSION: The importance of a population-based approach to pediatric palliative care in a state that is geographically large and diverse like Queensland is highlighted. This article may provide valuable information to other health care providers who care for children with life-limiting illnesses.

Amino acid substitution in the active site of DNA polymerase ß explains the energy barrier of the nucleotidyl transfer reaction.

DNA polymerase ß (pol ß) is a bifunctional enzyme widely studied for its roles in base excision DNA repair, where one key function is gap-filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol ß in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3'group, thus activating O3'as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1000-fold less active than the wild-type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wild-type enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3'proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3' proton transfer during the reaction path are dramatically altered compared with wild-type. Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3'. The theoretical results with the wild-type enzyme indicate an early charge reorganization associated with the O3' proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site.

Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

A pilot study of the effectiveness of home teleconsultations in paediatric palliative care.

We conducted a pilot study to investigate the effectiveness of a home telehealth service for paediatric palliative care consultations. Over a 10 week period, 14 of the 17 caregivers approached to be part of the study agreed to participate. Families were allocated, non-randomly, to a control group (usual care) or an intervention group (usual care with the addition of home telehealth consultations). The primary outcome measure was quality-of-life score. Caregivers were surveyed for up to 99 days following recruitment. A descriptive analysis of the quality-of-life data showed no differences between caregivers in the two groups. However, important lessons were learnt regarding factors which influence the success of studies in this population group, and the domains of caregiver quality-of-life that warrant intervention. Palliative care is complex, and multiple interventions and supports are required if care is to be managed at home. Home telehealth consultations are a feasible and acceptable means of facilitating a palliative care consultation which can reduce the burden on families at a distressing time.

Weakly Antiferromagentic Coupling Via Superexchange Interaction Between Mn(II)-Mn(II) Atoms: A QM/MM Study of the Active Site of Human Cytosolic X-Propyl Aminopeptidase P.

We investigate the dinuclear manganese, Mn(II)-Mn(II), active site of human cytosolic X-propyl aminopeptidase (XPNPEP1) employing the QM/MM method. The optimized structure supports two manganese atoms at the active site and excludes the possibility of a single Mn(II) atom or other combination of divalent metal ions: Ca(II), Fe(II), Mg(II). A broken symmetry solution verifies an antiferromagnetically coupled state between the Mn(II)-Mn(II) pair, which is the ground state. From the energy difference between the high spin state (HS) and the broken symmetry state (BS), we estimate the exchange coupling constant, J, to be 5.15 cm(-1). Also, we observe multiple bridges (p orbitals) from solvent and two carboxylate linking to the Mn(II)-Mn(II), which leads to the weakly antiferromagnetic interaction of d(5)-d(5) electrons through superexchange coupling.