Structural differences between glycosylated, disulfide-linked heterodimeric Knob-into-Hole Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products.

An increasing number of bispecific therapeutic antibodies are progressing through clinical development. The Knob-into-Hole (KiH) technology uses complementary mutations in the CH3 region of the antibody Fc fragment to achieve heavy chain heterodimerization. Here we describe the X-ray crystal structures of glycosylated and disulfide-engineered heterodimeric KiH Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products. The heterodimer structure confirms the KiH design principle and supports the hypothesis that glycosylation stabilizes a closed Fc conformation. Both homodimer structures show parallel Fc fragment architectures, in contrast to recently reported crystal structures of the corresponding aglycosylated Fc fragments which in the absence of disulfide mutations show an unexpected antiparallel arrangement. The glycosylated Knob-Knob Fc fragment is destabilized as indicated by variability in the relative orientation of its CH3 domains. The glycosylated Hole-Hole Fc fragment shows an unexpected intermolecular disulfide bond via the introduced Y349C Hole mutation which results in a large CH3 domain shift and a new CH3-CH3 interface. The crystal structures of glycosylated, disulfide-linked KiH Fc fragment and its Knob-Knob and Hole-Hole side products reported here will facilitate further design of highly efficient antibody heterodimerization strategies.

Structure and assembly of the spliceosomal snRNPs. Novartis Medal Lecture.

The spliceosome is a macromolecular machine that carries out the excision of introns from eukaryotic pre-mRNAs and splicing together of exons. Four large RNA-protein complexes, called the U1, U2, U4/U6 and U5 small nuclear ribonucleoprotein particles (snRNPs), and some non-snRNP proteins assemble around three short conserved sequences within the intron in an ordered manner to form the active spliceosome. We aim to provide insight into the molecular details of the mechanism of pre-mRNA splicing through crystallographic studies of the snRNPs. We have solved the X-ray crystal structure of some snRNP proteins as part of either protein-protein complexes or RNA-protein complexes. These structures have provided an important insight into the overall architecture of the U1 and U2 snRNPs and the mechanisms of RNA-protein and protein-protein recognition.

X-ray structure analyses of photosynthetic reaction center variants from Rhodobacter sphaeroides: structural changes induced by point mutations at position L209 modulate electron and proton transfer.

The structures of the reaction center variants Pro L209 --> Tyr, Pro L209 --> Phe, and Pro L209 --> Glu from the photosynthetic purple bacterium Rhodobacter sphaeroides have been determined by X-ray crystallography to 2.6-2.8 A resolution. These variants were constructed to interrupt a chain of tightly bound water molecules that was assumed to facilitate proton transfer from the cytoplasm to the secondary quinone Q(B) [Baciou, L., and Michel, H. (1995) Biochemistry 34, 7967-7972]. However, the amino acid exchanges Pro L209 --> Tyr and Pro L209 --> Phe do not interrupt the water chain. Both aromatic side chains are oriented away from this water chain and interact with three surrounding polar side chains (Asp L213, Thr L226, and Glu H173) which are displaced by up to 2.6 A. The conformational changes induced by the bulky aromatic rings of Tyr L209 and Phe L209 lead to unexpected displacements of Q(B) compared to the wild-type protein. In the structure of the Pro L209 --> Tyr variant, Q(B) is shifted by approximately 4 A and is now located at a position similar to that reported for the wild-type reaction center after illumination [Stowell, M. H. B., et al. (1997) Science 276, 812-816]. In the Pro L209 --> Phe variant, the electron density map reveals an intermediate Q(B) position between the binding sites of the wild-type protein in the dark and the Pro L209 --> Tyr protein. In the Pro L209 --> Glu reaction center, the carboxylic side chain of Glu L209 is located within the water chain, and the binding site of Q(B) remains unchanged compared to the wild-type structure.

Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides reconstituted with anthraquinone as primary quinone Q(A).

In the photosynthetic reaction centre (RC) from the purple bacterium Rhodobacter sphaeroides, the primary quinone, a ubiquinone-10 (Q(A)), has been substituted by anthraquinone. Three-dimensional crystals have been grown from the modified RC and its structure has been determined by X-ray crystallography to 2.4 A resolution. The bindings of the head-group from ubiquinone-10 and of the anthraquinone ring are very similar. In particular, both rings are parallel to each other and the hydrogen bonds connecting the native ubiquinone-10 molecule to AlaM260 and HisM219 are conserved in the anthraquinone containing RC. The space of the phytyl tail missing in the anthraquinone exchanged RC is occupied by the alkyl chain of a detergent molecule. Other structural changes of the Q(A)-binding site are within the limit of resolution. Our structural data bring strong credit to the very large amount of spectroscopic data previously achieved in anthraquinone-replaced RCs and which have participated in the determination of the energetics of the quinone system in bacterial RCs.

Identification of a hydrogen bond in the phe M197-->Tyr mutant reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides by X-ray crystallography and FTIR spectroscopy.

In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X-ray structure analysis of the Phe M197-->Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 A resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al. [Biochemistry 32, 12875-12886, 1993] between the Tyr M197 hydroxy group and one of the 2a-acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the pi-conjugated 9-keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.