The Crystal Structure of Fragment Double-D from Cross-Linked Lamprey Fibrin Reveals Isopeptide Linkages across an Unexpected D−D Interface,

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Abstract
The crystal structure of fragment double-D from factor XIII-cross-linked lamprey fibrin has been determined at 2.9 Å resolution. The 180 kDa covalent dimer was cocrystallized with the peptide Gly-His-Arg-Pro-amide, which in many fibrinogens, but not that of lamprey, corresponds to the B-knob exposed by thrombin. The structure was determined by molecular replacement, a recently determined structure of lamprey fragment D being used as a search model. GHRPam was found in both the γ- and β-chain holes. Unlike the situation with fragment D, the crystal packing of the cross-linked double-D structure exhibits two different D−D interfaces, each γ-chain facing γ-chains on two other molecules. One of these (interface I) involves the asymmetric interface observed in all other D fragments and related structures. The other (interface II) encompasses a completely different set of residues. The two abutments differ in that interface I results in an “in line” arrangement of abutting molecules and the interface II in a “zigzag” arrangement. So far as can be determined (the electron density could only be traced on one side of the cross-links), it is the γ-chains of the newly observed zigzag units (interface II) that are joined by the reciprocal ε-amino-γ-glutamyl cross-links. Auspiciously, the same novel D−D interface was observed in two lower-resolution crystal structures of human double-D preparations that had been crystallized under unusual circumstances. These observations show that double-D structures are linked in a way that is sufficiently flexible to accommodate different D−D interfaces under different circumstances.