Developing Adnectins that target SRC co-activator binding to PXR: a structural approach toward understanding promiscuity of PXR.

The human pregnane X receptor (PXR) is a promiscuous nuclear receptor that functions as a sensor to a wide variety of xenobiotics and regulates expression of several drug metabolizing enzymes and transporters. We have generated "Adnectins", derived from 10th fibronectin type III domain ((10)Fn3), that target the PXR ligand binding domain (LBD) interactions with the steroid receptor co-activator-1 (SRC-1) peptide, displacing SRC-1 binding. Adnectins are structurally homologous to the immunoglobulin superfamily. Three different co-crystal structures of PXR LBD with Adnectin-1 and CCR1 (CC chemokine receptor-1) antagonist Compound-1 were determined. This structural information was used to modulate PXR affinity for a related CCR1 antagonist compound that entered into clinical trials for rheumatoid arthritis. The structures of PXR with Adnectin-1 reveal specificity of Adnectin-1 in not only targeting the interface of the SRC-1 interactions but also engaging the same set of residues that are involved in binding of SRC-1 to PXR. Substituting SRC-1 with Adnectin-1 does not alter the binding conformation of Compound-1 in the ligand binding pocket. The structure also reveals the possibility of using Adnectins as crystallization chaperones to generate structures of PXR with compounds of interest.

High-throughput screening of chromatographic separations: III. Monoclonal antibodies on ceramic hydroxyapatite.

High-throughput screening (HTS) of chromatography resins for identifying optimal protein purification conditions is becoming an integral part of industrial process development. In this work, ceramic hydroxyapatite (cHA) chromatography of 15 humanized monoclonal antibodies (mAbs) was examined by HTS. MAb binding, as quantified by partition coefficient (K(p)), was measured under 92 combinations of sodium chloride, phosphate, and pH. Binding varied inversely with these variables for all mAbs tested. However, the magnitudes of binding among mAbs under identical conditions varied significantly, showing a >1.5 log range in K(p). Analysis of variance (ANOVA) techniques were used to describe the binding of each mAb as a function of the three screen variables. Linear models relating log K(p) to the pH, log[sodium chloride], and log[phosphate] fit the data for each antibody with 93-96% accuracy. From these models, characteristic charge values for the cation exchange and metal coordination components of the multi-modal mAb/cHA interaction varied twofold across the mAbs, reflecting inherent variability in the number of contacts between a particular mAb and the cHA surface. Furthermore, we reduced the number of test conditions required from 92 to 8 while maintaining an accurate representation of the full binding response surface. This eight-point modeling method accurately predicted the binding behavior of mAbs as well as mAb aggregates, a common impurity in crude mAb preparations. Using this eight-point modeling method, binding and selectivity information for mAb and aggregate can be obtained from less than two milligrams of protein, making the method attractive for early manufacturability assessments.

A virus-virus interaction circumvents the virus receptor requirement for infection by pathogenic retroviruses.

During ongoing C-type retrovirus infection, the probability of leukemia caused by insertional gene activation is markedly increased by the emergence of recombinant retroviruses that repeatedly infect host cells. The murine mink cell focus-inducing (MCF) viruses with this property have acquired characteristic changes in the N-terminal domain of their envelope glycoprotein that specify binding to a different receptor than the parental ecotropic virus. In this report, we show that MCF virus infection occurs through binding to this receptor (termed Syg1) and, remarkably, by a second mechanism that does not utilize the Syg1 receptor. By the latter route, the N-terminal domain of the ecotropic virus glycoprotein expressed on the cell surface in a complex with its receptor activates the fusion mechanism of the MCF virus in trans. The rate of MCF virus spread through a population of permissive human cells was increased by establishment of trans activation, indicating that Syg1 receptor-dependent and -independent pathways function in parallel. Also, trans activation shortened the interval between initial infection and onset of cell-cell fusion associated with repeated infection of the same cell. Our findings indicate that pathogenic retrovirus infection may be initiated by virus binding to cell receptors or to the virus envelope glycoprotein of other viruses expressed on the cell surface. Also, they support a broader principle: that cooperative virus-virus interactions, as well as virus-host interactions, shape the composition and properties of the retrovirus quasispecies.

Structure and mechanism of a coreceptor for infection by a pathogenic feline retrovirus.

Infection of T lymphocytes by the cytopathic retrovirus feline leukemia virus subgroup T (FeLV-T) requires FeLIX, a cellular coreceptor that is encoded by an endogenous provirus and closely resembles the receptor-binding domain (RBD) of feline leukemia virus subgroup B (FeLV-B). We determined the structure of FeLV-B RBD, which has FeLIX activity, to a 2.5-A resolution by X-ray crystallography. The structure of the receptor-specific subdomain of this glycoprotein differs dramatically from that of Friend murine leukemia virus (Fr-MLV), which binds a different cell surface receptor. Remarkably, we find that Fr-MLV RBD also activates FeLV-T infection of cells expressing the Fr-MLV receptor and that FeLV-B RBD is a competitive inhibitor of infection under these conditions. These studies suggest that FeLV-T infection relies on the following property of mammalian leukemia virus RBDs: the ability to couple interaction with one of a variety of receptors to the activation of a conserved membrane fusion mechanism. A comparison of the FeLV-B and Fr-MLV RBD structures illustrates how receptor-specific regions are linked to conserved elements critical for postbinding events in virus entry.