Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I/II distinction of CD20 antibodies.

CD20 is a cell-surface marker of normal and malignant B cells. Rituximab, a monoclonal antibody targeting CD20, has improved the treatment of malignant lymphomas. Therapeutic CD20 antibodies are classified as either type I or II based on different mechanisms of killing malignant B cells. To reveal the molecular basis of this distinction, we fine-mapped the epitopes recognized by both types. We also determined the first X-ray structure of a type II antibody by crystallizing the obinutuzumab (GA101) Fab fragment alone and in complex with a CD20 cyclopeptide. Despite recognizing an overlapping epitope, GA101 binds CD20 in a completely different orientation than type I antibodies. Moreover, the elbow angle of GA101 is almost 30° wider than in type I antibodies, potentially resulting in different spatial arrangements of 2 CD20 molecules bound to a single GA101 or rituximab molecule. Using protein tomography, different CD20 complexes were found to be associated with the 2 antibodies, and confocal microscopy showed different membrane compartmentalization of these subpopulations of the cellular CD20 pool. Our findings offer a possible molecular explanation for the different cellular responses elicited by type I and II antibodies.

Visualization of SV2A conformations in situ by the use of Protein Tomography.

The synaptic vesicle protein 2A (SV2A), the brain-binding site of the anti-epileptic drug levetiracetam (LEV), has been characterized by Protein Tomography. We identified two major conformations of SV2A in mouse brain tissue: first, a compact, funnel-structure with a pore-like opening towards the cytoplasm; second, a more open, V-shaped structure with a cleft-like opening towards the intravesicular space. The large differences between these conformations suggest a high degree of flexibility and support a valve-like mechanism consistent with the postulated transporter role of SV2A. These two conformations are represented both in samples treated with LEV, and in saline-treated samples, which indicates that LEV binding does not cause a large-scale conformational change of SV2A, or lock a specific conformational state of the protein. This study provides the first direct structural data on SV2A, and supports a transporter function suggested by sequence homology to MFS class of transporter proteins.

The antibody zalutumumab inhibits epidermal growth factor receptor signaling by limiting intra- and intermolecular flexibility.

The epidermal growth factor receptor (EGFR) activates cellular pathways controlling cell proliferation, differentiation, migration, and survival. It thus represents a valid therapeutic target for treating solid cancers. Here, we used an electron microscopy-based technique (Protein Tomography) to study the structural rearrangement accompanying activation and inhibition of native, individual, EGFR molecules. Reconstructed tomograms (3D density maps) showed a level of detail that allowed individual domains to be discerned. Monomeric, resting EGFR ectodomains demonstrated large flexibility, and a number of distinct conformations were observed. In contrast, ligand-activated EGFR complexes were detected only as receptor dimers with ring-like conformations. Zalutumumab, a therapeutic inhibitory EGFR antibody directed against domain III, locked EGFR molecules into a very compact, inactive conformation. Biochemical analyses showed bivalent binding of zalutumumab to provide potent inhibition of EGFR signaling. The structure of EGFR-zalutumumab complexes on the cell surface visualized by Protein Tomography indicates that the cross-linking spatially separates the EGFR molecules' intracellular kinase domains to an extent that appears incompatible with the induction of signaling. These insights into the mechanisms of action of receptor inhibition may also apply to other cell-surface tyrosine kinase receptors of the ErbB family.

Characterization of recombinant soluble macrophage scavenger receptor MARCO.

MARCO is a type II transmembrane protein of the class A scavenger receptor family. It has a short N-terminal cytoplasmic domain, a transmembrane domain, and a large extracellular part composed of a 75-residue long spacer domain, a 270-residue collagenous domain, and a 99-residue long scavenger receptor cysteine-rich (SRCR) domain. Previous studies have indicated a role for this receptor in anti-microbial host defense functions. In this work we have produced the extracellular part of MARCO as a recombinant protein, and analyzed its binding properties. The production of this protein, soluble MARCO (sMARCO), has made it possible for the first time to study MARCO and its binding properties in a cell-free system. Using circular dichroism analyses, a protease-sensitive assay, and rotary shadowing electron microscopy, sMARCO was shown to have a triple-helical collagenous structure. Rotary shadowing also demonstrated that the molecules often associate with each other via the globes. sMARCO was found to bind avidly both heat-killed and living bacteria. Lipopolysaccharide, an important component of the outer membrane of Gram-negative bacteria, was shown to be a ligand of MARCO. Studies with different bacterial strains indicated that the O-side chain of lipopolysaccharide is not needed for the bacterial recognition. Finally, the C-terminal SRCR domain was also produced as a recombinant protein, and its bacteria-binding capability was studied. Although the transfection experiments with transmembrane MARCO variants have indicated a crucial role for this domain in bacterial binding, the monomeric domain exhibited low, barely detectable bacteria-binding activity. Thus, it is possible that cooperation between the SRCR domain and the collagenous domain is needed for high-affinity bacterial binding, or that the SRCR domain has to be in a trimeric form to effectively bind to bacteria.

Arginine residues in domain V have a central role for bacteria-binding activity of macrophage scavenger receptor MARCO.

MARCO is a bacteria-binding macrophage-specific scavenger receptor that plays a role in innate immune response. MARCO has short intracellular and transmembrane domains, as well as a large extracellular domain composed of a spacer domain, a long collagenous domain, and a C-terminal scavenger receptor cysteine-rich domain (SRCR), domain V. As yet, no specific function has been assigned to the SRCR domain of scavenger receptors. In the present study, we generated several human and mouse MARCO variants with deletions or single amino acid substitutions and localized the primary bacteria-binding region to domain V. Furthermore, analysis of the MARCO variants containing only portions of domain V demonstrated a crucial role for an arginine-rich segment for this function. More precisely, the motif RXR was identified as an essential element for high-affinity bacterial binding. The results indicate that the binding properties of MARCO differ from those of the other class A scavenger receptors, SR-A and SRCL, whose ligand-binding function has been localized to the collagenous domain.