Preston Hensley

Crystal Structure of the Extracellular Domain from P0, the Major Structural Protein of Peripheral Nerve Myelin

P0, the major protein of peripheral nerve myelin, mediates membrane adhesion in the spiral wraps of the myelin sheath. We have determined the crystal structure of the extracellular domain from P0 (P0ex) at 1.9 A resolution. P0ex is folded like a typical immunoglobulin variable-like domain; five residues at the C-terminus are disordered, suggesting a flexible linkage to the membrane. The requirements for crystallization of P0ex are similar to those for maintaining the native extracellular spacing of adjacent myelin lamellae; thus, given the self-adhesive character of P0ex, the crystal itself may reveal some of the natural interactions that occur between P0 molecules in myelin. The structure leads to the suggestion that P0 extracellular domains may emanate from the membrane surface as tetramers that link to tetramers on the opposing membrane surface, to result in the formation of networks of molecules. We report analytical ultracentrifugation data for P0ex that support this idea.

Herpesvirus Entry Mediator Ligand (HVEM-L), a Novel Ligand for HVEM/TR2, Stimulates Proliferation of T Cells and Inhibits HT29 Cell Growth

Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-κB and AP-1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF-related proteins derived from an expressed sequence tag data base. One of these, which we designated HVEM-L, specifically bound to HVEM-Fc with an affinity of 44 nm. This association was confirmed with soluble and membrane forms of both receptor and ligand. HVEM-L mRNA is expressed in spleen, lymph nodes, macrophages, and T cells and encodes a 240-amino acid protein. A soluble, secreted form of the protein stimulates proliferation of T lymphocytes during allogeneic responses, inhibits HT-29 cell growth, and weakly stimulates NF-κB-dependent transcription.

Kinetic analysis of estrogen receptor/ligand interactions

Surface plasmon resonance biosensor technology was used to directly measure the binding interactions of small molecules to the ligand-binding domain of human estrogen receptor. In a screening mode, specific ligands of the receptor were easily discerned from nonligands. In a high-resolution mode, the association and dissociation phase binding responses were shown to be reproducible and could be fit globally to a simple interaction model to extract reaction rate constants. On average, antagonist ligands (such as tamoxifen and nafoxidine) were observed to bind to the receptor with association rates that were 500-fold slower than agonists (such as estriol and β-estradiol). This finding is consistent with these antagonists binding to an altered conformation of the receptor. The biosensor assay also could identify subtle differences in how the same ligand interacted with two different isoforms of the receptor (α and β). The biosensors ability to determine kinetic rate constants for small molecule/protein interactions provides unique opportunities to understand the mechanisms associated with complex formation as well as new information to drive the optimization of drug candidates.

Binding Interactions of Human Interleukin 5 with Its Receptor α Subunit LARGE SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL STUDIES OF DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS

Human interleukin 5 (hIL5) and soluble forms of its receptor α subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor α subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-Å resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor α subunit (shIL5Rα). Additionally, the relative abundance of ligand and receptor in the hIL5·shIL5Rα complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 n M, respectively, for the binding of hIL5 to shIL5Rα and to a chimeric form of the receptor containing shIL5Rα fused to the immunoglobulin Fc domain (shIL5Rα-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kdvalues from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor α subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5Rα in Drosophila membranes was found to be 6 n M, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the α receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5·receptor αβ complex and signal transduction.

Determination of rate and equilibrium binding constants for macromolecular interactions by surface plasmon resonance

Publisher Summary This chapter focuses on the determination of rate and equilibrium binding constants for macromolecular interactions by surface plasmon resonance. The characterization of the kinetics and thermodynamics of macromolecular interactions is increasingly important for developing an understanding of the molecular basis of events such as cell adhesion and viral infection, and it may ultimately aid in the rational design of antagonists of such interactions. Surface plasmon resonance (SPR) detectors, such as the BIAcore instrument, Pharmacia, allow for the direct visualization of these macromolecular interactions in real time, and thus the data obtained contains information on the rate and equilibrium binding constants that describe the interaction being investigated. SPR is an optical phenomenon that occurs as a result of the total internal reflection (TIR) of light at a metal film-liquid interface. TIR is observed in situations where light travels through an optically dense medium such as glass and is reflected back through that medium at the interface with a less optically dense medium such as buffer, provided the angle of incidence is greater than the critical angle required for the pair of optical media.

Determining confidence intervals for parameters derived from analysis of equilibrium analytical ultracentrifugation data.

In the above discussion, we have introduced the profiling approach of Bates and Watts. It is an easy to implement, empirical approach to the determination of confidence intervals for parameters in nonlinear models. We have applied the approach to the analysis of equilibrium sedimentation data and have demonstrated that, although models for analyzing such data are formally nonlinear they are functionally linear. As such, linear approximation confidence intervals for the parameters are adequate for these models and data sets. Further, we have been able to examine the effect of implementing a multiple independent variable approach (in this case, using multiple rotor speeds) on the precision of the analysis. We found that the standard errors of the parameters were reduced and that this is accounted for by either the increase in the number of data points or the decreases in parameter correlation. In this case, profiling helped to visualize the effect on the sum of squares surface of reducing parameter correlation, making the effect of the small decreases in the correlation of some parameters more evident. Using profiling, it should be easy to explore other methods for the improvement of the analysis of ultracentrifugation data and to be able to quantitate the improvement. With the above discussion as an example, it is likely that the profiling approach should be quite useful and broadly applicable in the analysis of data in terms of nonlinear models.

Secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus DNA polymerase

The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial α-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional 1H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal α-helices separated by a less ordered region. A series of “alanine scan” peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.

Measurement of protein interaction bioenergetics: Application to structural variants of anti-sCD4 antibody

This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.

Evaluating energetics of erythropoietin ligand binding to homodimerized receptor extracellular domains

Abstract A number of techniques have been employed to characterize the energetics of EPO-EPOR-Fc interactions. AUC studies have shown that EPO and EPOR-Fc exist as monomers at concentrations less that 10 μM. Under these conditions, EPO and the EPOR-Fc associate to form a 1:1 complex and this complex does not undergo any further assembly processes. Studies in which the biological activity of EPO at a cell surface is competed by free and dimerized receptor show that the dimerized receptor is 750-fold more potent. This suggests that EPO is bound by both receptor subunits on the Fc chimera, as shown in Fig. 9D. This assembly model provides a foundation for interpretation of the kinetic, thermodynamic, and spectral results. SPR kinetic analyses of the EPO-EPOR-Fc interaction yields association and dissociation rate constants of 8.0 × 10 7 M −1 sec −1 and 2.4 × 10 −4 sec −1 , respectively, for an overall affinity of 3 p M (see Fig. 12). The half-maximal response in a cellular proliferation assay is evoked at an EPO concentration of 10 p M , 54 which is similar to the affinity kinetically determined for the EPOR-Fc. This value suggests that the EPOR-Fc chimera may be a reasonable model for the receptor on a cell surface (see Fig. 17). The use of this reagent is also supported by the studies of Remy et al. , who demonstrate that the EPOR is likely to exist as a dimer on the cell surface, in the absence of ligand. Titration calorimetry confirms the 1:1 stoichiometry, observed by AUC and SPR approaches. Further, the temperature dependence of the enthalpy yields a heat capacity that can be interpreted in terms of a large conformational change in the EPOR on EPO binding. Comparing the structures of the free and complexed receptor, some conformational changes are noted in loops L3 and L6. 18 However, these changes are small compared with the conformational changes predicted from an analysis of the calorimetric data reported here, i.e., equivalent to the folding of ∼70 amino acids. The change in buried surface area between the free and complexed EPOR, determined from structural data, is also quite small when compared with the predicted value of 7500 A 2 from calorimetry. Further studies need to be done to rationalize these observations. These may include an attempt to determine if conformational changes are communicated to the Fc domain and the extent to which EPOR extracellular domains are oriented on the Fc domain in a manner that faithfully reflects their orientation on a cell surface. Finally, while changes in the CD spectra are observed on binding of EPO to the EPOR-Fc, and the monomeric receptor, these changes may be due to subtle changes in the microenvironments of tryptophans and tyrosines and do not require conformational changes of the magnitude suggested from the calorimetry results. In summary, to define macromolecular interactions in solution, the stoichiometry, thermodynamics, and kinetics of assembly need to be understood. This task requires that a multitechnology approach be implemented. Here, AUC established an assembly model and provided a foundation on which SPR, ITC, and CD studies could be based and from which interpretation of these data could be extended. SPR established that the affinity of the dimerized receptor was high and ITC suggested that there may be a significant conformational change on binding. CD suggested that observed spectral changes may be due to these presumed conformational changes, but would also be consistent with more subtle changes. These studies further demonstrate that the EPOR-Fc is a valid model for the dimerized receptor on the cell surface and, as such, will be a useful tool for probing the differences in the interactions of the receptor dimer with EPO agonists and antogonists.

Experimental Dissection of Protein-Protein Interactions in Solution

Publisher Summary Protein–protein interactions play crucial roles in a wide variety of processes, which define cell structure and function. This chapter describes the emerging tools and strategies for determining the kinetics and thermodynamics of protein–protein interactions. These biophysical methods can be used to play dominant roles as core tools for studying the structural and functional molecular properties of protein–protein interactions. In general, these methods require relatively small amounts (milligrams) of protein material and are universally applicable to the study of proteins. Although biophysical methods are capable of providing molecular-level details of protein–protein interactions, the increase in resolution to the molecular level is accompanied by an increased need to validate interpretation of the data. The chapter discusses the synergistic benefits of using multiple independent methods.