Aglycosylated knob and hole Fc homodimers are anti-parallel

Bispecific antibody and antibody-like molecules are of wide interest as potential therapeutics that can recognize two distinct targets. Among the variety of ways such molecules have been engineered is by creating ""knob"" and ""hole"" heterodimerization sites in the CH3 domains of two antibody heavy chains. The molecules produced in this manner maintain their biological activities while differing very little from the native human IgG sequence. To better understand the knob-into-hole interface, the molecular mechanism of heterodimerization, and to engineer Fc domains that could improve the assembly and purity of bispecific antibodies, we sought crystal structures of heterodimeric and homodimeric aglycosylated Fc fragments bearing ""knob"" and ""hole"" mutations. The structure of the knob-into-hole Fc was determined at 2.64Å. Except for the sites of mutation, the structure is very similar to that of the native human IgG1 Fc, consistent with a hetero-dimer interaction kinetic KD <1 nM. Homodimers of the ""knob"" and ""hole"" mutants were also obtained and their X-ray structures were determined at resolutions of 2.5Å and 2.1Å, respectively. Both kinds of homodimers adopt a head-to-tail quaternary structure and thus do not contain direct knob-knob or hole-hole CH3 interactions. By adding site-directed mutations at F241 and F243 in the CH2 domains, the head-to-tail arrangement was disfavored, leading to increases in both the rate and efficiency of bispecific (heterodimer) assembly.

Crystallographic studies for the folding of an extending peptide

Full-length proteins can fold into thermodynamically stable structures at an exceptionally fast rate as shown by in vitro experiments. In contrast, it takes much more time to finish nascent protein folding than full-length protein folding, because nascent protein folding depends on the rate of ribosome biosynthesis in the living cell. Therefore nascent polypeptide chains in vivo fold co-translationally in different manners from the full-length proteins. However, the transient structures and the co-translational folding pathway are not well understood. In order to reveal the atomic details of nascent protein folding, we studied the hPin1 WW domain, which consists of two beta-hairpins between the three-stranded beta-sheets. Here we report a series of WW domain N-terminal fragment structures with increasing amino acid length by using circular dichroism spectroscopy and X-ray crystallography. In crystallization, maltose-binding protein was fused just behind the WW domain fragments to fix the C-terminus as nascent proteins are anchored to the ribosome. Co-translational folding of beta-sheet-rich proteins is discussed based on our finding that intermediate-length fragments unexpectedly take a helical conformation, even though the full-length protein has no helical regions. Furthermore, in a region of one of the loop structures of the full-length protein, these fragments take different formations. Our results suggest that the newly synthesized polypeptides adopt the most stable conformation during the course of peptide extension and fold into the native structures, eventually.

Ultra-high resolution structure of high-potential iron-sulfur protein

It is important for understanding the electron transfer reaction to include the information about valence shell electrons and hydrogen atoms into crystal structure refinement. High-potential iron-sulfur protein (HiPIP) possesses a Fe4S4 cluster which exhibits +2/+3 redox states and acts as an electron carrier from cytochrome bc1 complex to the reaction center complex in photosynthetic purple bacteria. We have reported the X-ray crystal structure of HiPIP from Thermochtomatium tepidum at 0.72 Å resolution (1). Recently, we have successfully collected 0.48 Å resolution data of HiPIP using high-energy X-rays (31 keV) in BL41XU beamline of SPring-8. We performed multipolar refinement with the MoPro program (2) to consider valence shell electrons in the structure refinement of HiPIP. Refinement of multipolar parameters was applied to atoms of single conformational residues, water molecules with two hydrogen atoms, and the Fe4S4 cluster. After multipolar refinement, the deformation map clearly displays the distribution of valence shell electrons such as lone-pair electrons of carbonyl oxygen atoms, bonding electrons in aromatic rings, and d-orbital electrons of Fe atoms in the Fe4S4 cluster. The deformation map also indicates electrostatic interactions between the S atoms of Fe4S4-(Cys-Sγ)4 and protein environment. In addition, we performed preliminary neutron diffraction experiment at iBIX beamline of Japan Proton Accelerator Research Complex (J-PARC) and observed diffraction spots up to 1.17 Å resolution using HiPIP crystal with the size of 2.3 mm3. In the multipolar refinement, the positions of hydrogen atoms were fixed to the standard bond distances derived from neutron crystal structures of small molecules and atomic displacement parameters of hydrogen atoms were constrained to 1.2 or 1.5 fold of their root atoms. Therefore, a high resolution neutron structure of HiPIP will improve the results obtained from the multipolar refinement.