Zachary Johnson

Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 Å

Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes. Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes. A wide range of nucleoside-derived drugs, including anticancer drugs (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes. Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 Å. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes.

Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters

Concentrative nucleoside transporters (CNTs) are responsible for cellular entry of nucleosides, which serve as precursors to nucleic acids and act as signaling molecules. CNTs also play a crucial role in the uptake of nucleoside-derived drugs, including anticancer and antiviral agents. Understanding how CNTs recognize and import their substrates could not only lead to a better understanding of nucleoside-related biological processes but also the design of nucleoside-derived drugs that can better reach their targets. Here, we present a combination of X-ray crystallographic and equilibrium-binding studies probing the molecular origins of nucleoside and nucleoside drug selectivity of a CNT from Vibrio cholerae. We then used this information in chemically modifying an anticancer drug so that it is better transported by and selective for a single human CNT subtype. This work provides proof of principle for utilizing transporter structural and functional information for the design of compounds that enter cells more efficiently and selectively. DOI: http://dx.doi.org/10.7554/eLife.03604.001

Observations of a Synechococcus-dominated cyclonic eddy in open-oceanic waters of the Arabian Sea

The optical, chemical and biological characteristics of a cyclonic eddy were investigated in the Arabian Sea during November 1995. This eddy was 3,000 km2 in area and located 350 km offshore of the coast of Oman. The mixed layer of this feature extended to a depth of 17 m, below which oxygen concentrations were depleted to 10 percent of surface values. Chlorophyll a concentrations within the mixed layer averaged 1.7 mg m-3. Microscopic observations and flow cytometric measurements revealed that the algal community was dominated by the coccoid cyanobacterium Synechococcus. Detailed pigment analyses documented additional phytoplankton biomass contributions by Prochlorococcus, diatoms, dinoflagellates, pyrmnesiophytes, cryptophytes, chlorophytes and pelagophytes. This algal community caused a marked attenuation of the blue to bluegreen wavelengths of light, resulting in a preferential transmittance of green light with increasing depth. Measurements of photosynthetic performance and the spectral absorption coefficient document that the phytoplankton community was photo- and chromatically-adapted to the light environment within the eddy. The results of this field work support a previous laboratory study which found that the nonphotosynthetic carotenoid zeaxanthin produces significant decreases in the maximum quantum yield of photosynthesis of the marine cyanobacterium Synechococcus.

Ground truthing modeled kPAR and on deck primary productivity incubations with in situ observations

The Arabian Sea Process Study carried out on six cruises in 1995 provided observations that make possible the ground truthing of model estimates of kPAR and on deck productivity incubations with direct kPAR observations and in situ productivity incubations. THe Morel 1988 optical model did a good job of reproducing the observed values. Comparison of on deck and in situ depth profiles suggested that on deck incubators received more irradiance across the entire light gradient. This subsidy in E0 apparently came from efficient absorption of reflected light and low angle sunlight by small incubators. When depths were recalculated taking into account the irradiance subsidy, profiles of productivity as a function of depth determined by on deck incubations were essentially identical to in situ profiles.

Visualizing multistep elevator-like transitions of a nucleoside transporter

Membrane transporters move substrates across the membrane by alternating access of their binding sites between the opposite sides of the membrane. An emerging model of this process is the elevator mechanism, in which a substrate-binding transport domain moves a large distance across the membrane. This mechanism has been characterized by a transition between two states, but the conformational path that leads to the transition is not yet known, largely because the available structural information has been limited to the two end states. Here we present crystal structures of the inward-facing, intermediate, and outward-facing states of a concentrative nucleoside transporter from Neisseria wadsworthii. Notably, we determined the structures of multiple intermediate conformations, in which the transport domain is captured halfway through its elevator motion. Our structures present a trajectory of the conformational transition in the elevator model, revealing multiple intermediate steps and state-dependent conformational changes within the transport domain that are associated with the elevator-like motion.

Liposome Reconstitution and Transport Assay for Recombinant Transporters

Secondary active transporters are responsible for the cellular uptake of many biologically important molecules, including neurotransmitters, nutrients, and drugs. Because of their physiological and clinical importance, a method for assessing their transport activity in vitro is necessary to gain a better understanding of how these transporters function at the molecular level. In this chapter, we describe a protocol for reconstituting the concentrative nucleoside transporter from Vibrio cholerae into proteoliposomes. We then describe a radiolabeled substrate uptake assay that can be used to functionally characterize the transporter. These methods are relatively common and can be applied to other secondary active transporters, with or without some modification.

Primary production and irradiance during an intermonsoon cruise to the Arabian Sea (November, 1995)

The Process-6 cruise, of the Arabian Sea Expedition of the Joint Global Ocean Flux Study, occurred during an intermonsoon, in October-November 1995. A variety of water column conditions were sampled in terms of euphotic zone depths, autotrophic biomass, resident phytoplankton populations, and nutrients. We use measurements of the rate of primary production, quantities of chlorophyll a, the properties of phytoplankton absorption, daily time course of submarine PAR, and estimates of quantum yield to examine the variability seen. Both phytoplankton absorption and variations in quantum yields contribute to variations in primary production. Offshore populations exhibit a reduction in the apparent maximum quantum yield.

Modeled inherent scattering properties of small light-limited phytoplankton: implications for deep phytoplankton size class distributions

Small phytoplankton, ubiquitous throughout the worlds oceans, numerically dominate many open ocean ecosystems with increasing importance towards the base of the euphotic zone. As an example, light-limited deep secondary chlorophyll maxima are usually dominated by small phytoplankton species. Theoretical models describing light-particle interactions predict that small particles scatter light less efficiently than their larger counterparts. To investigate a possible relationship between the dominance of small phytoplankton in light-limited situations and efficiency predictions, a light scattering efficiency model based on Mie theory as approximated by Van de Hulst is used to determine scattering efficiency as a function of size. This scattering efficiency model, which approximates light-phytoplankton interactions by considering phytoplankton as homogeneous spheres, is driven by the spectral light field from an observed deep phytoplankton population dominated by small phytoplankton. This deep secondary chlorophyll maximum is discussed as an example of a highly efficient small phytoplankton population at the threshold of the euphotic zone which could benefit as a result of its size distribution.