An immunoglobulin light chain dimer with a large generic binding cavity was used as a host molecule for designing a series of peptide guest ligands. In a screening procedure peptides coupled to solid supports were systematically tested for binding activity by enzyme linked immunosorbent assays (ELISA). Key members of the binding series were synthesized in milligram quantities and diffused into crystals of the host molecule for X-ray analyses. These peptides were incrementally increased in size and affinity until they nearly filled the cavity. Progressive changes in binding patterns were mapped by comparisons of crystallographically refined structures of 14 peptide-protein complexes at 2.7 A resolution. These comparisons led to guidelines for ligand design and also suggested ways to modify previously established binding patterns. By manipulating equilibria involving histidine, for example, it was possible to abolish one important intramolecular interaction of the bound ligand and substitute another. These events triggered a change in conformation of the ligand from a compact to an extended form and a comprehensive change in the mode of binding to the protein. In dipeptides of histidine and proline, protonation of both imidazolium nitrogen atoms was used to program an end-to-end reversal of the direction in which the ligand was inserted into the binding cavity. Peptides cocrystallized with proteins produced complexes somewhat different in structure from those in which ligands were diffused into preexisting crystals. In such a large and malleable cavity, space utilization was thus different when a ligand was introduced before the imposition of crystal packing restraints.
Light (L) chain dimers expressed by multiple myeloma cells and collected as Bence‐Jones proteins from the urine of human subjects were tested for their ability to form deposits in fibroblast monolayer cell cultures. Bence‐Jones proteins from subjects with primary amyloidosis associated with L chains were shown to form fibrillar deposits by the in vitro assay introduced in this report. Filaments interspersed with nascent collagen could be detected after only 48 h. Deposition of L chains continued over a period of 72 h culminating in the appearance of dense fibrils with widths of 80–100 Å and a variety of lengths. Formation of amyloid‐like fibrils was accompanied by interference with the maturation of the collagen produced by the fibroblast cells. Fibrils composed of the Mcg λ‐type L chain were deposited between collagen fibers, thus expanding them laterally and leading to their partial disintegration. Mature collagen was completely missing from fibroblast monolayers exposed to the Sea λ chain and the Jen κ chain. Collagen with the characteristic striped pattern matured normally in control samples, such as those not dosed with amyloid precursors or those treated with a non‐amyloidogenic Bence‐Jones protein (e.g., the Hud λ chain dimer). By immunochemical techniques using fluorescein‐ and gold‐labeled anti‐L chain antibodies, amyloidogenic L chains were shown to decorate the strands of nascent collagen. This observation suggests that amyloidogenic L chains are concentrated in the extracellular matrix by monovalent antigen–antibody type reactions. The capacity of the Mcg L chain dimer to bind collagen‐derived sequences was tested by soaking crystals with a collagenase substrate, PZ‐Pro‐Leu‐Gly‐Pro‐D‐Arg. Difference Fourier analysis at 2.7 Å resolution indicated that the PZ‐peptide is a site‐filling ligand. It could not be removed from the active site by perfusion of the crystal with ammonium sulfate crystallizing media. Similar experiments with the collagen‐derived peptide (Pro‐Pro‐Gly)5 showed substantial hysteresis effects extending from one end of the Mcg dimer to the other. After the ligand was withdrawn, the active site of the Mcg dimer could no longer bind the PZ‐peptide. However, if the active site was first blocked by the PZ‐peptide and subsequently exposed to the (Pro‐Pro‐Gly)5 peptide, the difference Fourier map was indistinguishable from that obtained with the PZ‐peptide alone. We concluded that amyloidogenic L chains such as the Mcg dimer could be concentrated in the perivascular space by binding to normal tissue constituents. These components include nascent collagen, which can be deterred from maturing as a result of this binding. Participation in such pathological activity is also self‐destructive to the amyloidogenic L chains, which lose their binding capabilities for collagen‐derived peptides and also become susceptible to irreversible conversion to amyloid fibrils. All of these events may be prevented by prior treatment of the amyloidogenic L chains with site‐filling ligands. Copyright © 20...
Complexes of nucleotides, peptides and aromatic hapten-like compounds with immunoglobulin fragments were studied by X-ray analysis. After tri- or hexanucleotides of deoxythymidylate were diffused into triclinic crystals of a Fab (BV04-01) with specificity for single-stranded DNA, extensive changes were detected throughout the structure of the protein. The Fab co-crystallized with a tri- or pentanucleotide in a different space group (monoclinic), an observation sometimes correlated with alterations in the structure of the 'native' protein. Structural analyses of the co-crystals are in progress for direct comparisons with the unliganded Fab. In crystals of a human (Mcg) Bence-Jones dimer, synthetic opioid peptides, chemotactic peptides or dinitrophenyl (DNP) derivatives could be diffused into a large conical binding cavity. The conformations of both the ligand and the protein were usually altered during the binding process. At the base of the cavity tyrosine residues could be displaced like trap-doors to permit entry of some opioid peptides and DNP compounds into a deep binding pocket. In co-crystals of the dimer and bis(DNP)lysine, two ligand molecules were bound in tandem, one in the main cavity and the second in the deep pocket. One ligand adopted an extended conformation, with the epsilon-DNP ring near the floor of the main cavity and the alpha-DNP group in solvent outside the binding site. There were no significant conformational changes in the protein. In contrast, the second ligand was very compact, with DNP rings immersed in the deep pocket, and the binding site was expanded to accommodate the oversized ligand. Peptides designed to be specific for the main cavity were incrementally constructed from minimal binding units by M. Geysen, G. Trippick, S. Rodda and their colleagues. A pentapeptide optimized for binding by this method was diffused into a crystal of the dimer and found by Fourier difference analysis to lodge exclusively in the main cavity as predicted. Binding regions in the BV04-01 Fab and the Mcg dimer were markedly different in size and shape. The Fab had a groove-type site, in which a layer of sidechains acted like a false floor over regions analogous to the cavity and deep pocket of the Bence-Jones dimer.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.