Protein-protein recognition: The neonatal Fe receptor and immunoglobulin G

The neonatal Fe receptor (FeRn) binds the Fe portion of immunoglobulin G (IgG) at the acidic pH of endosomes or the gut and releases IgG at the alkaline pH of blood. FeRn is responsible for the maternofetal transfer of IgG and for rescuing endocytosed IgG from a default degradative pathway. We investigated how FeRn interacts with IgG by constructing a heterodimeric form of the Fe (hdFc) that contains one FeRn binding site. This molecule was used to characterize the interaction between one FeRn molecule and one Fe and to determine under what conditions FeRn forms a dimer. The hdFc binds one FeRn molecule at pH 6.0 with a K_d of 80 nM. In solution and with FeRn anchored to solid supports, the heterodimeric Fe does not induce a dimer of FeRn molecules. FcRnhdFc complex crystals were obtained and the complex structure was solved to 2.8 Å resolution. Analysis of this structure refined the understanding of the mechanism of the pH-dependent binding, shed light on the role played by carbohydrates in the Fe binding, and provided insights on how to design therapeutic IgG antibodies with longer serum half-lives. The FcRn-hdFc complex in the crystal did not contain the FeRn dimer. To characterize the tendency of FeRn to form a dimer in a membrane we analyzed the tendency of the hdFc to induce cross-phosphorylation of FeRn-tyrosine kinase chimeras. We also constructed FeRn-cyan and FeRn-yellow fluorescent proteins and have analyzed the tendency of these molecules to exhibit fluorescence resonance energy transfer. As of now, neither of these analyses have lead to conclusive results. In the process of acquiring the context to appreciate the structure of the FcRn-hdFc interface, we developed a study of 171 other nonobligate protein-protein interfaces that includes an original principal component analysis of the quantifiable aspects of these interfaces.

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.