Functional tuning of the catalytic residue pKa in a de novo designed esterase

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p‐nitrophenyl acetate (pNPA) and 4‐nitrophenyl‐(2‐phenyl)‐propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single‐histidine residue (His¹⁴⁴) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK_a value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK_a calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK_a value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK_a value of this residue in water. We determined that a short‐range favorable interaction with Glu¹²⁷ contributes to the elevated pK_a of His¹⁴⁴. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK_a of its active nucleophile, His¹⁴⁴, by 0.7 units. As a direct result of the decrease in the His¹⁴⁴ pK_a value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine‐promoted catalysis. Proteins 2017; 85:1656–1665. © 2017 Wiley Periodicals, Inc.     

The abundance of an invasive freshwater snail Tarebia granifera (Lamarck, 1822) in the Nseleni River,South Africa

The invasive freshwater snail Tarebia granifera (Lamarck, 1822) was first reported in South Africa in 1999 and it has become widespread across the country, with some evidence to suggest that it reduces benthic macroinvertebrate biodiversity. The current study aimed to identify the primary abiotic drivers behind abundance patterns of T. granifera, by comparing the current abundance of the snail in three different regions, and at three depths, of the highly modified Nseleni River in KwaZulu-Natal, South Africa. Tarebia granifera was well established throughout the Nseleni River system, with an overall preference for shallow waters and seasonal temporal patterns of abundance. Although it is uncertain what the ecological impacts of the snail in this system are, its high abundances suggest that it should be controlled where possible and prevented from invading other systems in the region.     

Extended polypeptide linkers establish the spatial architecture of a pyruvate dehydrogenase multienzyme complex

Icosahedral pyruvate dehydrogenase (PDH) enzyme complexes are molecular machines consisting of a central E2 core decorated by a shell of peripheral enzymes (E1 and E3) found localized at a distance of approximately 75-90 A from the core. Using a combination of biochemical, biophysical, and cryo-electron microscopic techniques, we show here that the gap between the E2 core and the shell of peripheral enzymes is maintained by the flexible but extended conformation adopted by 60 linker polypeptides that radiate outwards from the inner E2 core, irrespective of the E1 or E3 occupancy. The constancy of the gap is thus not due to protein-protein interactions in the outer protein shell. The extended nature of the E2 inner-linker regions thereby creates the restricted annular space in which the lipoyl domains of E2 that carry catalytic intermediates shuttle between E1, E2, and E3 active sites, while their conformational flexibility facilitates productive encounters.     

The resolution of five racemic alpha-lactams by HPLC

The resolution of five racemic alpha-lactams (1a-d,g) using HPLC is reported. Five different Pirkle-type stationary phases were tested. The enantiomers of alpha-lactams containing the trityl group (1a-d) were separated (selectivity factors ranging from 1.08 to 1.20) using a mobile phase of hexane/2-propanol:98/2 and a stationary phase consisting of the 3,5-dinitroaniline derivative of (S)-valine with a urea linkage. Among the dialkyl-substituted alpha-lactams (1e-g), only 1,3-di-tert-butylaziridinone (1g) could be resolved, but only partially (selectivity factor = 1.07), with a mobile phase of hexane/1,2-dichloroethane:95/5 and the stationary phase consisting of the 3,5-dinitrobenzoic acid derivative of (R)-1-naphthylglycine.     

Patterns of host ant use by sympatric populations of <Emphasis Type="Italic">Maculinea alcon</Emphasis> and <Emphasis Type="Italic">M. ‘rebeli’</Emphasis> in the Carpathian Basin

Maculinea butterflies show social parasitism via obligatory myrmecophily as their larvae are adopted and raised to pupation by Myrmica ants. Suitable hosts differ for different Maculinea species, and host ant specificity can further differ at the population-level. Although early studies suggested single ant species as main hosts for each Maculinea species, it has recently become clear that their host ant specificity is more complex. Maculinea alcon and Maculinea ‘rebeli’ have variously been separated according to adult and larval morphology, phenology, and their use of different ecosystems, including host plant and host ant species. However, recent genetic evidence has questioned their separation as good species. Here we compare the use of host ants by M. alcon and M. ‘rebeli’ at the regional scale in NE-Hungary and Transylvania (Romania), where molecular studies have found no species-level separation between the two forms. We opened 778 nests of Myrmica ants and searched for Maculinea specimens (larvae, pupae and exuviae) shortly before imago emergence from the nest in seven M. alcon sites, six M. ‘rebeli’- sites and one site where both M. alcon and M. ‘rebeli’ are syntopic. In all, Maculinea caterpillars were found in the nests of seven different ant species (M. alcon was recorded mainly with Myrmica scabrinodis and occasionally with M. salina and M. vandeli; M. ‘rebeli’ used mainly M. scabrinodis, M. sabuleti and M. schencki and occasionally M. lonae and M. specioides). Myrmica scabrinodis was found to be a general host of both M. alcon and M. ‘rebeli’, which is the first record for a common host ant of these two closely related butterflies within the same region. However there were also differences in host ant use patterns between the sites occupied by the two Maculinea taxa, which reflect differences in Myrmica communities between the two types of habitat. Possible explanations for the similar but not identical host use patterns of M. alcon and M. ‘rebeli’, and their relevance for the question of whether they are separate species are discussed. Received 27 November 2007; revised 28 May 2008; accepted 11 June 2008.     

Implementation of an insecticide-treated net subsidy scheme under a public-private partnership for malaria control in Tanzania – challenges in implementation

Background

In the past decade there has been increasing visibility of malaria control efforts at the national and international levels. The factors that have enhanced this scenario are the availability of proven interventions such as artemisinin-based combination therapy, the wide scale use of insecticide-treated nets (ITNs) and a renewed emphasis in indoor residual house-spraying. Concurrently, there has been a window of opportunity of financial commitments from organizations such as the Global Fund for HIV/AIDS, Tuberculosis and Malaria (GFATM), the President's Malaria Initiative and the World Bank Booster programme.

Methods

The case study uses the health policy analysis framework to analyse the implementation of a public-private partnership approach embarked upon by the government of Tanzania in malaria control – 'The Tanzania National Voucher Scheme'- and in this synthesis, emphasis is on the challenges faced by the scheme during the pre-implementation (2001 – 2004) and implementation phases (2004 – 2005). Qualitative research tools used include: document review, interview with key informants, stakeholder's analysis, force-field analysis, time line of events, policy characteristic analysis and focus group discussions. The study is also complemented by a cross-sectional survey, which was conducted at the Rufiji Health Demographic Surveillance Site, where a cohort of women of child-bearing age were followed up regarding access and use of ITNs.

Results

The major challenges observed include: the re-introduction of taxes on mosquito nets and related products, procurement and tendering procedures in the implementation of the GFATM, and organizational arrangements and free delivery of mosquito nets through a Presidential initiative.

Conclusion

The lessons gleaned from this synthesis include: (a) the consistency of the stakeholders with a common vision, was an important strength in overcoming obstacles, (b) senior politicians often steered the policy agenda when the policy in question was a 'crisis event', the stakes and the visibility were high, (c) national stakeholders in policy making have an advantage in strengthening alliances with international organizations, where the latter can become extremely influential in solving bottlenecks as the need arises, and (d) conflict can be turned into an opportunity, for example the Presidential initiative has inadvertently provided Tanzania with important lessons in the organization of 'catch-up' campaigns.     

The Role of Ongoing Dendritic Oscillations in Single-Neuron Dynamics

The dendritic tree contributes significantly to the elementary computations a neuron performs while converting its synaptic inputs into action potential output. Traditionally, these computations have been characterized as both temporally and spatially localized. Under this localist account, neurons compute near-instantaneous mappings from their current input to their current output, brought about by somatic summation of dendritic contributions that are generated in functionally segregated compartments. However, recent evidence about the presence of oscillations in dendrites suggests a qualitatively different mode of operation: the instantaneous phase of such oscillations can depend on a long history of inputs, and under appropriate conditions, even dendritic oscillators that are remote may interact through synchronization. Here, we develop a mathematical framework to analyze the interactions of local dendritic oscillations and the way these interactions influence single cell computations. Combining weakly coupled oscillator methods with cable theoretic arguments, we derive phase-locking states for multiple oscillating dendritic compartments. We characterize how the phase-locking properties depend on key parameters of the oscillating dendrite: the electrotonic properties of the (active) dendritic segment, and the intrinsic properties of the dendritic oscillators. As a direct consequence, we show how input to the dendrites can modulate phase-locking behavior and hence global dendritic coherence. In turn, dendritic coherence is able to gate the integration and propagation of synaptic signals to the soma, ultimately leading to an effective control of somatic spike generation. Our results suggest that dendritic oscillations enable the dendritic tree to operate on more global temporal and spatial scales than previously thought; notably that local dendritic activity may be a mechanism for generating on-going whole-cell voltage oscillations.     

Zinc binding to the Tyr402 and His402 allotypes of complement factor H: possible implications for age-related macular degeneration

The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD. Heterozygous FH is inhibited by zinc, which causes FH to aggregate. Here, zinc binding to homozygous FH was studied. By analytical ultracentrifugation, large amounts of oligomers were observed with both the native Tyr402 and the AMD-risk His402 homozygous allotypes of FH and both the recombinant SCR-6/8 allotypes with Tyr/His402. X-ray scattering also showed that both FH and SCR-6/8 allotypes strongly aggregated at > 10 μM zinc. The SCR-1/5 and SCR-16/20 fragments were less likely to bind zinc. These observations were supported by bioinformatics predictions. Starting from known zinc binding sites in crystal structures, we predicted 202 putative partial surface zinc binding sites in FH, most of which were in SCR-6. Metal site prediction web servers also suggested that SCR-6 and other domains bind zinc. Predicted SCR-6/8 dimer structures showed that zinc binding sites could be formed at the protein-protein interface that would lead to daisy-chained oligomers. It was concluded that zinc binds weakly to FH at multiple surface locations, most probably within the functionally important SCR-6/8 domains, and this explains why zinc inhibits FH activity. Given the high pathophysiological levels of bioavailable zinc present in subretinal deposits, we discuss how zinc binding to FH may contribute to deposit formation and inflammation associated with AMD.     

Human and rhesus APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H demonstrate a conserved capacity to restrict Vif-deficient HIV-1

Successful intracellular pathogens must evade or neutralize the innate immune defenses of their host cells and render the cellular environment permissive for replication. For example, to replicate efficiently in CD4(+) T lymphocytes, human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral infectivity factor (Vif) that promotes pathogenesis by triggering the degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3 proteins have been implicated in HIV-1 restriction, but the relevant repertoire remains ambiguous. Here we present the first comprehensive analysis of the complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction. In addition to APOBEC3G, we find that three other human APOBEC3 proteins, APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors. These four proteins are expressed in CD4(+) T lymphocytes, are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, and they are all neutralized by the simian immunodeficiency virus Vif protein. On the other hand, neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant impact on HIV-1 replication. These data strongly implicate a combination of four APOBEC3 proteins--APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H--in HIV-1 restriction.