James W. Bryson

Protein Design: A Hierarchic Approach

The de novo design of peptides and proteins has recently emerged as an approach for investigating protein structure and function. Designed, helical peptides provide model systems for dissecting and quantifying the multiple interactions that stabilize secondary structure formation. De novo design is also useful for exploring the features that specify the stoichiometry and stability of α-helical coiled coils and for defining the requirements for folding into structures that resemble native, functional proteins. The design process often occurs in a series of discrete steps. Such steps reflect the hierarchy of forces required for stabilizing tertiary structures, beginning with hydrophobic forces and adding more specific interactions as required to achieve a unique, functional protein.

Native-like and structurally characterized designed α-helical bundles

Abstract A number of coiled coils and α-helical bundles have recently been designed, and many have now been structurally characterized by X-ray crystallography. Others have not been as well characterized structurally but exhibit native-like properties in aqueous solution. Both areas of investigation have contributed greatly to our understanding of the nature of specificity in this class of molecules.

Correlation of High-Throughput Pregnane X Receptor (PXR) Transactivation and Binding Assays

Pregnane X receptor (PXR) transactivation and binding assays have been developed into high-throughput assays, which are robust and reproducible (Z′ > 0.5). For most compounds, there was a good correlation between the results of the transactivation and binding assays. EC50 values of compounds in the transactivation assay correlated reasonably well with their IC50 values in the binding assay. However, there were discrepancies with some compounds showing high binding affinity in the binding assay translated into low transactivation. The most likely cause for these discrepancies was an agonist-dependent relationship between binding affinity and transactivation response. In general, compounds that bound to human PXR and transactivated PXR tended to be large hydrophobic molecules.

Engineering of a Novel Anti-CD40L Domain Antibody for Treatment of Autoimmune Diseases

CD40–CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a “domain Ab” (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb–Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb–Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin–induced Ab responses, alloantigen-induced T cell proliferation, “heart-to-ear” transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.

A Monovalent Anti-Human CD28 Domain Antibody Antagonist: Preclinical Efficacy and Safety

Targeting the CD28-CD80/86 pathway with an anti-CD28 antagonist is a promising alternative to current therapies for autoimmunity. However, attempts at generating conventional anti-CD28 mAbs lacking stimulatory activity has been challenging. In this study, we describe anti-human CD28 receptor antagonist domain Abs (dAbs) that are specific for human CD28. These dAbs are potent inhibitors of T cell activation, with an EC50 of 35 ± 14 ng/ml for inhibition of proliferation. The EC50 of 53 ± 11 ng/ml in an ex vivo CD28 receptor occupancy assay corresponds with in vitro functional activity, suggesting a direct correlation. The anti-CD28 dAb is equipotent in the inhibition of CD80- and CD86-mediated T cell proliferation and does not interfere with CTLA-4–mediated downmodulation of CD86 expression on APCs. The anti-CD28 dAbs are monomeric and do not demonstrate any evidence of agonism or costimulatory activity. In cynomolgus monkeys, the anti-CD28 dAb demonstrated pharmacodynamic activity, as measured by the inhibition of a T cell–dependent Ab response, without evidence of T cell depletion or cytokine release. Furthermore, there was a strong correlation between systemic exposure, duration, and extent of CD28 receptor occupancy, and pharmacodynamic activity. Taken together, these data support clinical evaluation of this novel anti-CD28 dAb for the treatment of autoimmune diseases.

De novo design of heterotrimeric coiled coils.

The three‐helix bundle is a common structural motif among natural proteins. It has been observed in numerous important proteins, such as fibrinogen, laminin, spectrin, dystrofin, hemagglutinin, and mannose binding proteins. The three‐helix bundle is a simple structure in which three α‐helices pack against each other, with a slight left‐handed twist. Because of its simplicity relative to other structural motifs, the three‐helix bundle can be conveniently used both to clarify the forces responsible for the protein folding and stability, and for the design of novel proteins. In this paper we describe the design, synthesis, and characterization of three peptides that self‐assemble into antiparallel, heterotrimeric coiled coils. The experimental results, obtained from CD spectroscopy and ultracentrifugation equilibrium sedimentation, indicate that the mixture of the three peptides preferentially forms heterotrimers; moreover, these aggregates represent attractive systems for combinatorial design of libraries of pseudo C3 symmetric ligands or binding sites.

Chemical and Genetic Studies of Copper Resistance in E. coli

Copper is an essential trace element required for bacterial growth, but is toxic at high levels of free ions. Bacteria are thus presented with the complex problem of obtaining and storing sufficient quantities of copper for normal function of several enzymes while, on die other hand, being able to survive when confronted with concentrations of copper that exceed a toxic threshold. The molecular mechanisms of metal ion detoxification are well understood only for a few systems, most notably for mercury resistance.1,2 For recent reviews of a number of bacterial metal resistance systems, see Plasmid, Vol. 27(1), 1992 and reference 3. The discovery of plasmid-borne copper resistance in bacteria has provided accessible systems for genetic and phenomenological study of copper metabolism. Study of these extrachromosomal systems has recently provided the impetus and the methods for identification of chromosomally-encoded copper homeostasis systems in Pseudomonas syringae pv. tomato (P. syringae)4,5,6 and in Echerichia coli (E. coli) 7,8,9 The two best characterized copper resistance systems are the plasmid-encoded systems cop in P. syringae and pco in E. coli. These two determinants are remarkably similar genetically, but as will be shown they differ fundamentally in the mechanism of copper resistance. This paper will focus first on the genetics and regulation of copper resistance in the cop and pco systems, then on the chemical mechanisms of copper resistance.

Functional Antagonism of Human CD40 Achieved by Targeting a Unique Species-Specific Epitope.

Current clinical anti-CD40 biologic agents include both antagonist molecules for the treatment of autoimmune diseases and agonist molecules for immuno-oncology, yet the relationship between CD40 epitope and these opposing biological outcomes is not well defined. This report describes the identification of potent antagonist domain antibodies (dAbs) that bind to a novel human CD40-specific epitope that is divergent in the CD40 of nonhuman primates. A similarly selected anti-cynomolgus CD40 dAb recognizing the homologous epitope is also a potent antagonist. Mutagenesis, biochemical, and X-ray crystallography studies demonstrate that the epitope is distinct from that of CD40 agonists. Both the human-specific and cynomolgus-specific molecules remain pure antagonists even when formatted as bivalent Fc-fusion proteins, making this an attractive therapeutic format for targeting hCD40 in autoimmune indications.

Cloning, purification, crystallization and preliminary X-ray analysis of the catalytic domain of human receptor-like protein tyrosine phosphatase γ in three different crystal forms.

Protein tyrosine phosphatase γ is a membrane-bound receptor and is designated RPTPγ. RPTPγ and two mutants, RPTPγ(V948I, S970T) and RPTPγ(C858S, S970T), were recombinantly expressed and purified for X-ray crystallographic studies. The purified enzymes were crystallized using the hanging-drop vapor-diffusion method. Crystallographic data were obtained from several different crystal forms in the absence and the presence of inhibitor. In this paper, a description is given of how three different crystal forms were obtained that were used with various ligands. An orthorhombic crystal form and a trigonal crystal form were obtained both with and without ligand, and a monoclinic crystal form was only obtained in the presence of a particularly elaborated inhibitor.

Time-Resolved Limited Proteolysis of Mitogen-Activated Protein Kinase-Activated Protein Kinase-2 Determined by LC/MS Only

Mass spectrometry has gained prominence in limited proteolysis studies largely due to its unparalleled precision in determining protein molecular mass. However, proteolytic fragments usually cannot be identified through direct mass measurement, since multiple subsequences of a protein can frequently be matched to observed masses of proteolytic fragments. Therefore, additional information from N-terminal sequencing is often needed. Here we demonstrate that mass spectrometry analysis of the time course of limited proteolysis reactions provides new information that is self-sufficient to identify all proteolytic fragments. The method uses a non-specific protease like subtilisin and exploits information contained in the time-resolved dataset such as: increased likelihood of identifying larger fragments generated during initial proteolysis solely by their masses, additivity of the masses of two mutually exclusive sequence regions that generate the full-length molecule (or an already assigned subfragment), and analyses of the proteolytic subfragment patterns that are facilitated by having established the initial cleavage sites. We show that the identities of the observed proteolytic fragments can be determined by LC/MS alone because enough constraints exist in the time-resolved dataset. For a medium-sized protein, it takes about 8 h to complete the study, a significant improvement over the traditional SDS-PAGE and N-terminal sequencing method, which usually takes several days. We illustrate this method with application to the catalytic domain of mitogen-activated protein kinase-activated protein kinase-2, and compare the results with N-terminal sequencing data and the known X-ray crystal structure.