Structure-, stereochemistry-, and metal-regulated DNA binding/cleaving molecules

In the cell, the binding of proteins to specific sequences of double helical DNA is essential for controlling the processes of protein synthesis (at the level of DNA transcription) and cell proliferation (at the level of DNA replication). In the laboratory, the sequence-specific DNA binding/cleaving properties of restriction endonuclease enzymes (secreted by microorganisms to protect them from foreign DNA molecules) have helped to fuel a revolution in molecular biology. The strength and specificity of a protein:DNA interaction depend upon structural features inherent to the protein and DNA sequences, but it is now appreciated that these features (and therefore protein:DNA complexation) may be altered (regulated) by other protein:DNA complexes, or by environmental factors such as temperature or the presence of specific organic molecules or inorganic ions. It is also now appreciated that molecules much smaller than proteins (including antibiotics of molecular weight less than 2000 and oligonucleotides) can bind to double-helical DNA in sequence-specific fashion. Elucidation of structural motifs and microscopic interactions responsible for the specific molecular recognition of DNA leads to greater understanding of natural processes and provides a basis for the design of novel sequence-specific DNA binding molecules. This thesis describes the synthesis and DNA binding/cleaving characteristics of molecules designed to probe structural, stereochemical, and environmental factors that regulate sequence-specific DNA recognition.

Chapter One introduces the DNA minor groove binding antibiotics Netropsin and Distamycin A, which are di- and tri(N-methylpyrrolecarboxamide) peptides, respectively. The method of DNA affinity cleaving, which has been employed to determine DNA binding properties of designed synthetic molecules is described. The design and synthesis of a series of Netropsin dimers linked in tail-to-tail fashion (by oxalic, malonic, succinic, or fumaric acid), or in head-to-tail fashion (by glycine, β-alanine, and γ-aminobutanoic acid (Gaba)) are presented. These Bis(Netropsin)s were appended with the iron-chelating functionality EDTA in order to make use of the technique of DNA affinity cleaving. Bis(Netropsin)-EDTA compounds are analogs of penta(N-methylpyrrolecarboxamide)-EDTA (P5E), which may be considered a head-to-tail Netropsin dimer linked by Nmethylpyrrolecarboxamide. Low- and high-resolution analysis of pBR322 DNA affinity cleaving by the iron complexes of these molecules indicated that small changes in the length and nature of the linker had significant effects on DNA binding/cleaving efficiency (a measure of DNA binding affinity). DNA binding/cleaving efficiency was found to decrease with changes in the linker in the order β-alanine > succinamide > fumaramide > N-methylpyrrolecarboxamide > malonamide >glycine, γ-aminobutanamide > oxalamide. In general, the Bis(Netropsin)-EDTA:Fe compounds retained the specificity for seven contiguous A:T base pairs characteristic of P5E:Fe binding. However, Bis(Netropsin)Oxalamide- EDTA:Fe exhibited decreased specificity for A:T base pairs, and Bis(Netropsin)-Gaba-EDT A:Fe exhibited some DNA binding sites of less than seven base pairs. Bis(Netropsin)s linked with diacids have C₂-symmmetrical DNA binding subunits and exhibited little DNA binding orientation preference. Bis(Netropsin)s linked with amino acids lack C₂-symmetrical DNA binding subunits and exhibited higher orientation preferences. A model for the high DNA binding orientation preferences observed with head-to-tail DNA minor groove binding molecules is presented.

Chapter Two describes the design, synthesis, and DNA binding properties of a series of chiral molecules: Bis(Netropsin)-EDTA compounds with linkers derived from (R,R)-, (S,S)-, and (RS,SR)-tartaric acids, (R,R)-, (S,S)-, and (RS,SR)-tartaric acid acetonides, (R)- and (S)-malic acids, N ,N-dimethylaminoaspartic acid, and (R)- and (S)-alanine, as well as three constitutional isomers in which an N-methylpyrrolecarboxamide (P1) subunit and a tri(N-methylpyrrolecarboxamide)-EDTA (P3-EDTA) subunit were linked by succinic acid, (R ,R)-, and (S ,S)-tartaric acids. DNA binding/cleaving efficiencies among this series of molecules and the Bis(Netropsin)s described in Chapter One were found to decrease with changes in the linker in the order β-alanine > succinamide > P1-succinamide-P3 > fumaramide > (S)-malicamide > N-methylpyrrolecarboxamide > (R)-malicamide > malonamide > N ,N-dimethylaminoaspanamide > glycine = Gaba = (S,S)-tartaramide = P1-(S,S)-tanaramide-P3 > oxalamide > (RS,SR)-tartaramide = P1- (R,R)-tanaramide-P3 > (R,R)-tartaramide (no sequence-specific DNA binding was detected for Bis(Netropsin)s linked by (R)- or (S)-alanine or by tartaric acid acetonides). The chiral molecules retained DNA binding specificity for seven contiguous A:T base pairs. From the DNA affinity cleaving data it could be determined that: 1) Addition of one or two substituents to the linker of Bis(Netropsin)-Succinamide resulted in stepwise decreases in DNA binding affinity; 2) molecules with single hydroxyl substituents bound DNA more strongly than molecules with single dimethylamino substituents; 3) hydroxyl-substituted molecules of (S) configuration bound more strongly to DNA than molecules of (R) configuration. This stereochemical regulation of DNA binding is proposed to arise from the inherent right-handed twist of (S)-enantiomeric Bis(Netropsin)s versus the inherent lefthanded twist of (R)-enantiomeric Bis(Netropsin)s, which makes the (S)-enantiomers more complementary to the right-handed twist of B form DNA.

Chapter Three describes the design and synthesis of molecules for the study of metalloregulated DNA binding phenomena. Among a series of Bis(Netropsin)-EDTA compounds linked by homologous tethers bearing four, five, or six oxygen atoms, the Bis(Netropsin) linked by a pentaether tether exhibited strongly enhanced DNA binding/cleaving in the presence of strontium or barium cations. The observed metallospecificity was consistent with the known affinities of metal cations for the cyclic hexaether 18-crown-6 in water. High-resolution DNA affinity cleaving analysis indicated that DNA binding by this molecule in the presence of strontium or barium was not only stronger but of different sequence-specificity than the (weak) binding observed in the absence of metal cations. The metalloregulated binding sites were consistent with A:T binding by the Netropsin subunits and G:C binding by a strontium or barium:pentaether complex. A model for the observed positive metalloregulation and novel sequence-specificity is presented. The effects of 44 different cations on DNA affinity cleaving by P5E:Fe were examined. A series of Bis(Netropsin)-EDTA compounds linked by tethers bearing two, three, four, or five amino groups was also synthesized. These molecules exhibited strong and specific binding to A:T rich regions of DNA. It was found that the iron complexes of these molecules bound and cleaved DNA most efficiently at pH 6.0-6.5, while P5E:Fe bound and cleaved most efficiently at pH 7.5-8.0. Incubating the Bis(Netropsin) Polyamine-EDTA:Fe molecules with K₂PdCl₄ abolished their DNA binding/cleaving activity. It is proposed that the observed negative metalloregulation arises from kinetically inert Bis(Netropsin) Polyamine:Pd(II) complexes or aggregates, which are sterically unsuitable for DNA complexation. Finally, attempts to produce a synthetic metalloregulated DNA binding protein are described. For this study, five derivatives of a synthetic 52 amino acid residue DNA binding/cleaving protein were produced. The synthetic mutant proteins carried a novel pentaether ionophoric amino acid residue at different positions within the primary sequence. The proteins did not exhibit significant DNA binding/cleaving activity, but they served to illustrate the potential for introducing novel amino acid residues within DNA binding protein sequences, and for the development of the tricyclohexyl ester of EDTA as a superior reagent for the introduction of EDT A into synthetic proteins.

Chapter Four describes the discovery and characterization of a new DNA binding/cleaving agent, [SalenMn(III)]OAc. This metal complex produces single- and double-strand cleavage of DNA, with specificity for A:T rich regions, in the presence of oxygen atom donors such as iodosyl benzene, hydrogen peroxide, or peracids. Maximal cleavage by [SalenMn(III)]OAc was produced at pH 6-7. A comparison of DNA singleand double-strand cleavage by [SalenMn(III)]⁺ and other small molecules (Methidiumpropyl-EDTA:Fe, Distamycin-EDTA:Fe, Neocarzinostatin, Bleomycin:Fe) is presented. It was found that DNA cleavage by [SalenMn(III)]⁺ did not require the presence of dioxygen, and that base treatment of DNA subsequent to cleavage by [SalenMn(III)]⁺ afforded greater cleavage and alterations in the cleavage patterns. Analysis of DNA products formed upon DNA cleavage by [SalenMn(III)] indicated that cleavage was due to oxidation of the sugar-phosphate backbone of DNA. Several mechanisms consistent with the observed products and reaction requirements are discussed.

Chapter Five describes progress on some additional studies. In one study, the DNA binding/cleaving specificities of Distamycin-EDTA derivatives bearing pyrrole N-isopropyl substituents were found to be the same as those of derivatives bearing pyrrole N-methyl substituents. In a second study, the design of and synthetic progress towards a series of nucleopeptide activators of transcription are presented. Five synthetic plasmids designed to test for activation of in vitro run-off transcription by DNA triple helix-forming oligonucleotides or nucleopeptides are described.

Chapter Six contains the experimental documentation of the thesis work.

I. Site effects and exciton structure in molecular crystals - Benzene. II. The first and second triplet states of solid benzene

The effect of intermolecular coupling in molecular energy levels (electronic and vibrational) has been investigated in neat and isotopic mixed crystals of benzene. In the isotopic mixed crystals of C₆H₆, C₆H₅D, m-C₆H₄D₂, p-C₆H₄D₂, sym-C₆H₃D₃, C₆D₅H, and C₆D₆ in either a C₆H₆ or C₆D₆ host, the following phenomena have been observed and interpreted in terms of a refined Frenkel exciton theory: a) Site shifts; b) site group splittings of the degenerate ground state vibrations of C₆H₆, C₆D₆, and sym-C₆H₃D₃; c) the orientational effect for the isotopes without a trigonal axis in both the ¹B_2u electronic state and the ground state vibrations; d) intrasite Fermi resonance between molecular fundamentals due to the reduced symmetry of the crystal site; and e) intermolecular or intersite Fermi resonance between nearly degenerate states of the host and guest molecules. In the neat crystal experiments on the ground state vibrations it was possible to observe many of these phenomena in conjunction with and in addition to the exciton structure.

To theoretically interpret these diverse experimental data, the concepts of interchange symmetry, the ideal mixed crystal, and site wave functions have been developed and are presented in detail. In the interpretation of the exciton data the relative signs of the intermolecular coupling constants have been emphasized, and in the limit of the ideal mixed crystal a technique is discussed for locating the exciton band center or unobserved exciton components. A differentiation between static and dynamic interactions is made in the Frenkel limit which enables the concepts of site effects and exciton coupling to be sharpened. It is thus possible to treat the crystal induced effects in such a fashion as to make their similarities and differences quite apparent.

A calculation of the ground state vibrational phenomena (site shifts and splittings, orientational effects, and exciton structure) and of the crystal lattice modes has been carried out for these systems. This calculation serves as a test of the approximations of first order Frenkel theory and the atom-atom, pair wise interaction model for the intermolecular potentials. The general form of the potential employed was V(r) = Be^-Cr - A/r⁶ ; the force constants were obtained from the potential by assuming the atoms were undergoing simple harmonic motion.

In part II the location and identification of the benzene first and second triplet states (³B_1u and ³E_1u) is given.

The connection between single species and ecosystems

Ecosystem level models are motivated by some combination of scientific and practical concerns. Those models motivated by practical concerns are likely to bear little historical relation to previous models. Mechanisms of interaction between particular species and their ecosystems vary enormously. Some species literally construct their own ecosystems. Others have more or less complex and important interactions with other species so that their presence or absence may alter the ecosystem. Prior information about the natural history of particular species can make ecosystem investigations quicker, cheaper, and more effective. The optimal resource for preparing to deal with the unlimited diversity of questions asked of ecologists would be a large' computerized databank of natural history observations for as many species as possible.

Uptake and intracytoplasmic storage of pigmented particles by human CD34+stromal cells/telocytes: endocytic property of telocytes

We studied the phagocytic-like capacity of human CD34+ stromal cells/telocytes (TCs). For this, we examined segments of the colon after injection of India ink to help surgeons localize lesions identified at endoscopy. Our results demonstrate that CD34+ TCs have endocytic properties (phagocytic-like TCs: phTCs), with the capacity to uptake and store India ink particles. phTCs conserve the characteristics of TCs (long, thin, bipolar or multipolar, moniliform cytoplasmic processes/telopodes, with linear distribution of the pigment) and maintain their typical distribution. Likewise, they are easily distinguished from pigment-loaded macrophages (CD68+ macrophages, with oval morphology and coarse granules of pigment clustered in their cytoplasm). A few c-kit/CD117+ interstitial cells of Cajal also incorporate pigment and may conserve the phagocytic-like property of their probable TC precursors. CD34+ stromal cells in other locations (skin and periodontal tissues) also have the phagocytic-like capacity to uptake and store pigments (hemosiderin, some components of dental amalgam and melanin). This suggests a function of TCs in general, which may be related to the transfer of macromolecules in these cells. Our ultrastructural observation of melanin-storing stromal cells with characteristics of TCs (telopodes with dichotomous branching pattern) favours this possibility. In conclusion, intestinal TCs have a phagocytic-like property, a function that may be generalized to TCs in other locations. This function (the ability to internalize small particles), together with the capacity of these cells to release extracellular vesicles with macromolecules, could close the cellular bidirectional cooperative circle of informative exchange and intercellular interactions.

Natural mortality rate, annual fecundity, and maturity at length for Greenland halibut (Reinhardtius hippoglossoides) from the northeastern Pacific Ocean

Mortality, fecundity, and size at maturity are important life history traits, and their interactions determine the evolution of life history strategies (Roff, 1992; Stearns, 1992; Charnov, 2002). These same traits are also important for population dynamics models (Hunter et al., 1992; Clark, 1999). It is increasingly important to accurately determine Greenland halibut (Reinhardtius hippoglossoides) life history traits and to correctly assess the status of its stocks because low recruitment or low biomass estimates have led to catch restrictions in the Bering Sea and Aleutian Islands (Ianelli et al.1), the Northeastern Arctic (Ådlandsvik et al., 2004), and the Northwest Atlantic (Bowering and Nedreaas, 2000).

PGPRs and nitrogen-fixing legumes: a perfect team for efficient Cd phytoremediation?

Cadmium (Cd) is a toxic, biologically non-essential and highly mobile metal that has become an increasingly important environmental hazard to both wildlife and humans. In contrast to conventional remediation technologies, phytoremediation based on legume rhizobia symbiosis has emerged as an inexpensive decontamination alternative which also revitalize contaminated soils due to the role of legumes in nitrogen cycling. In recent years, there is a growing interest in understanding symbiotic legume rhizobia relationship and its interactions with Cd. The aim of the present review is to provide a comprehensive picture of the main effects of Cd in N-2-fixing leguminous plants and the benefits of exploiting this symbiosis together with plant growth promoting rhizobacteria to boost an efficient reclamation of Cd-contaminated soils.

Holistic fisheries management: combining macroecology, ecology, and evolutionary biology

Ecosystem-based management is one of many indispensable components of objective, holistic management of human impacts on nonhuman systems. By itself, however, ecosystem-based management carries the same risks we face with other forms of current management; holism requires more. Combining single-species and ecosystem approaches represents progress. However, it is now recognized that management also needs to be evosystem-based. In other words, management needs to account for all coevolutionary and evolutionary interactions among all species; otherwise we fall far short of holism. Fully holistic practices are quite distinct from the approaches to the management of fisheries that are applied today. In this paper, we show how macroecological patterns can guide management consistently, objectively, and holistically. We present one particular macroecological pattern with two applications. The first application is a case study of fisheries from the Baltic Sea involving historical data for two species; the second involves a sample of 44 species of primarily marine fish worldwide. In both cases we evaluate historical fishing rates and determine holistic/systemic sustainable single-species fishing rates to illustrate that conventional fisheries management leads to much more extensive and pervasive overfishing than currently realized; harvests are, on average, over twenty-fold too large to be fully sustainable. In general, our approach involves not only the sustainability of fisheries and related resources but also the sustainability of the ecosystems and evosystems in which they occur. Using macroecological patterns accomplishes four important goals: 1) Macroecology becomes one of the interdisciplinary components of management. 2) Sustainability becomes an option for harvests from populations of individual species, species groups, ecosystems, and the entire marine environment. 3) Policies and goals are reality-based, holistic, or fully systemic; they account for ecological as well as evolutionary factors and dynamics (including management itself). 4) Numerous management questions can be addressed.

The design, construction, operation, and maintenance of the Replicated Modular Estuarine Mesocosm

Perhaps the most difficult job of the ecotoxicologist is extrapolating data calculated from laboratory experiments with high precision and accuracy into the real world of highly-dynamics aquatic environments. The establishment of baseline laboratory toxicity testing data for individual compounds and ecologically important and field studies serve as a precursor to ecosystem level studies needed for ecological risk assessment. The first stage in the field portion of risk assessment is the determination of actual environmental concentrations of the contaminant being studied and matching those concentrations with laboratory toxicity tests. Risk estimates can be produced via risk quotients that would determine the probability that adverse effects may occur. In this first stage of risk assessment, environmental realism is often not achieved. This is due, in part, to the fact that single-species laboratory toxicity tests, while highly controlled, do not account for the complex interactions (Chemical, physical, and biological) that take place in the natural environment. By controlling as many variables in the laboratory as possible, an experiment can be produced in such a fashion that real effects from a compound can be determined for a particular test organism. This type of approach obviously makes comparison with real world data most difficult. Conversely, field oriented studies fall short in the interpretation of ecological risk assessment because of low statistical power, lack of adequate replicaiton, and the enormous amount of time and money needed to perform such studies. Unlike a controlled laboratory bioassay, many other stressors other than the chemical compound in question affect organisms in the environment. These stressors range from natural occurrences (such as changes in temperature, salinity, and community interactions) to other confounding anthropogenic inputs. Therefore, an improved aquatic toxicity test that will enhance environmental realism and increase the accuracy of future ecotoxicological risk assessments is needed.

Monitoring well-being and changing environmental conditions in coastal communities: development of an assessment method

The intersection of social and environmental forces is complex in coastal communities. The well-being of a coastal community is caught up in the health of its environment, the stability of its economy, the provision of services to its residents, and a multitude of other factors. With this in mind, the project investigators sought to develop an approach that would enable researchers to measure these social and environmental interactions. The concept of well-being proved extremely useful for this purpose. Using the Gulf of Mexico as a regional case study, the research team developed a set of composite indicators to be used for monitoring well-being at the county-level. The indicators selected for the study were: Social Connectedness, Economic Security, Basic Needs, Health, Access to Social Services, Education, Safety, Governance, and Environmental Condition. For each of the 37 sample counties included in the study region, investigators collected and consolidated existing, secondary data representing multiple aspects of objective well-being. To conduct a longitudinal assessment of changing wellbeing and environmental conditions, data were collected for the period of 2000 to 2010. The team focused on the Gulf of Mexico because the development of a baseline of well-being would allow NOAA and other agencies to better understand progress made toward recovery in communities affected by the Deepwater Horizon oil spill. However, the broader purpose of the project was to conceptualize and develop an approach that could be adapted to monitor how coastal communities are doing in relation to a variety of ecosystem disruptions and associated interventions across all coastal regions in the U.S. and its Territories. The method and models developed provide substantial insight into the structure and significance of relationships between community well-being and environmental conditions. Further, this project has laid the groundwork for future investigation, providing a clear path forward for integrated monitoring of our nation’s coasts. The research and monitoring capability described in this document will substantially help counties, local organizations, as well state and federal agencies that are striving to improve all facets of community well-being.

A biogeographic asssessment of seabirds, deep sea corals and ocean habitats of the New York Bight: science to support offshore spatial planning

This report provides a compilation of new maps and spatial assessments for seabirds, bathymetry, surficial sediments, deep sea corals, and oceanographic habitats in support of offshore spatial planning led by the New York Department of State Ocean and Great Lakes Program. These diverse ecological themes represent priority information gaps left by past assessments and were requested by New York to better understand and balance ocean uses and environmental conservation in the Atlantic. The main goal of this report is to translate raw ecological, geomorphological and oceanographic data into maps and assessments that can be easily used and understood by coastal managers involved in offshore spatial planning. New York plans to integrate information in this report with other ecological, geophysical and human use data to obtain a broad perspective on the ocean environment, human uses and their interactions. New York will then use this information in an ecosystem-based framework to coordinate and support decisions balancing competing demands in their offshore environment, and ultimately develop a series of amendments to New York’s federally approved Coastal Management Program. The targeted users of this report and the compiled spatial information are New York coastal managers, but other State and federal decision-makers, offshore renewable energy development interests and environmental advocates will also find the information useful. In addition, the data and approaches will be useful to regional spatial planning initiatives set up by the Mid-Atlantic Regional Council on the Ocean (MARCO) and federal regional planning bodies for coastal and marine spatial planning.

The biology and ecology of the invasive Indo-Pacific lionfish

The Indo-Pacific lionfishes, Pterois miles and P. volitans, are now established along the Southeast U.S. and Caribbean and are expected to expand into the Gulf of Mexico and Central and South America. Prior to this invasion little was known regarding the biology and ecology of these lionfishes. I provide a synopsis of chronology, taxonomy, local abundance, reproduction, early life history and dispersal, venomology, feeding ecology, parasitology, potential impacts, and possible control and management strategies for the lionfish invasion. This information was collected by review of the literature and by direct field and experimental study. I confirm the existence of an unusual supraocular tentacle phenotype and suggest that the high prevalence of this phenotype in the Atlantic is not the result of selection, but likely ontogenetic change. To describe the trophic impacts of lionfish, I report a comprehensive assessment of diet that describes lionfish as a generalist piscivore that preys on over 40 species of teleost comprising more than 20 families. Next, I use the histology of gonads to describe both oogenesis and reproductive dynamics of lionfish. Lionfish mature relatively early and reproduce several times per month throughout the entire calendar year off North Carolina and the Bahamas. To investigate predation, an important component of natural mortality, I assessed the vulnerability of juvenile lionfish to predation by native serranids. Juvenile lionfish are not readily consumed by serranids, even after extreme periods of starvation. Last, I used a stage-based, matrix population model to estimate the scale of control that would be needed to reduce an invading population of lionfish. Together, this research provides the first comprehensive assessment on lionfish biology and ecology and explains a number of life history and ecological interactions that have facilitated the unprecedented and rapid establishment of this invasive finfish. Future research is needed to understand the scale of impacts that lionfish could cause, especially in coral reef ecosystems, which are already heavily stressed. This research further demonstrates the need for lionfish control strategies and more rigorous prevention and early detection and rapid response programs for marine non-native introductions.

Near and distant connection of atmospheric systems to ocean temperature change in the coastal California Current region

In studying hydrosphere, atmosphere, and biosphere interactions, it is useful to focus on specific subsystem processes and energy exchanges (forcing). Since subsystem scales range over ten orders of magnitude, it may be difficult to focus research on scales that will yield useful results in terms of establishing causal and predictive connections between more easily and less easily observed subsystems. In an effort to find pertinent scales, we have begun empirical investigations into relationships between atmospheric, oceanic, and biological systems having spatial scales exceeding 10^3 kilometers and temporal scales of six months or more.

Collective Cell Migration: Leadership, Invasion and Segregation

A number of biological processes, such as embryo development, cancer metastasis or wound healing, rely on cells moving in concert. The mechanisms leading to the emergence of coordinated motion remain however largely unexplored. Although biomolecular signalling is known to be involved in most occurrences of collective migration, the role of physical and mechanical interactions has only been recently investigated. In this paper, a versatile framework for cell motility is implemented in-silico in order to study the minimal requirements for the coordination of a group of epithelial cells. We find that cell motility and cell-cell mechanical interactions are sufficient to generate a broad array of behaviours commonly observed in vitro and in vivo. Cell streaming, sheet migration and susceptibility to leader cells are examples of behaviours spontaneously emerging from these simple assumptions, which might explain why collective effects are so ubiquitous in nature. This analysis provides also new insights into cancer metastasis and cell sorting, suggesting in particular that collective invasion might result from an emerging coordination in a system where single cells are mechanically unable to invade.

Feasibility study: the methods and approaches to implementing a SEREAD teacher training programme in BOBLME countries

Objectives included a desk-top feasibility study to explore opportunities to adapt the Scientific Educational Resources and Experience Associated with the Deployment of Argo profiling floats in the South Pacific Ocean (SEREAD) to BOBLME country schools.The programme included teacher resources on climate change and facilitating interactions between scientists, students and teachers.

Structural and functional characterization of the human CCR5 receptor in complex with HIV gp120 envelope glycoprotein and CD4 receptor by molecular modeling studies

The entry of human immunodeficiency virus (HIV) into cells depends on a sequential interaction of the gp120 envelope glycoprotein with the cellular receptors CD4 and members of the chemokine receptor family. The CC chemokine receptor CCR5 is such a receptor for several chemokines and a major coreceptor for the entry of R5 HIV type-1 (HIV-1) into cells. Although many studies focus on the interaction of CCR5 with HIV-1, the corresponding interaction sites in CCR5 and gp120 have not been matched. Here we used an approach combining protein structure modeling, docking and molecular dynamics simulation to build a series of structural models of the CCR5 in complexes with gp120 and CD4. Interactions such as hydrogen bonds, salt bridges and van der Waals contacts between CCR5 and gp120 were investigated. Three snapshots of CCR5-gp120-CD4 models revealed that the initial interactions of CCR5 with gp120 are involved in the negatively charged N-terminus (Nt) region of CCR5 and positively charged bridging sheet region of gp120. Further interactions occurred between extracellular loop2 (ECL2) of CCR5 and the base of V3 loop regions of gp120. These interactions may induce the conformational changes in gp120 and lead to the final entry of HIV into the cell. These results not only strongly support the two-step gp120-CCR5 binding mechanism, but also rationalize extensive biological data about the role of CCR5 in HIV-1 gp120 binding and entry, and may guide efforts to design novel inhibitors.