Vaheh Oganesyan

pH-dependent Binding Engineering Reveals an FcRn Affinity Threshold That Governs IgG Recycling

Background: FcRn controls the serum persistence of antibodies. Results: A panel of novel Fc mutations reveals sites controlling pH dependence and FcRn affinity. Conclusion: FcRn affinity thresholds determine IgG recycling efficiency. Significance: Knowledge of the relationship between FcRn binding and serum persistence can aid in designing better therapeutic antibodies. The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.

Mechanisms of neutralization of a human anti-α-toxin antibody.

Background: MEDI4893 is an anti-α-toxin (AT) antibody currently in clinical trial in the field of Staphylococcus aureus-mediated diseases. Results: Structure/function studies of MEDI4893 reveal its epitope and mechanisms of action. Conclusion: MEDI4893 recognizes a novel epitope and exhibits a possible dual neutralization mechanism. Significance: Understanding the molecular basis of AT/MEDI4893 interaction has important implications to design potent antibodies targeting Staphylococcus aureus. MEDI4893 is a neutralizing human monoclonal antibody that targets α-toxin (AT) and is currently undergoing evaluation in the field of Staphylococcus aureus-mediated diseases. We have solved the crystal structure of MEDI4893 Fab bound to monomeric AT at a resolution of 2.56 Å and further characterized its epitope using various engineered AT variants. We have found that MEDI4893 recognizes a novel epitope in the so-called “rim” domain of AT and exerts its neutralizing effect through a dual mechanism. In particular, MEDI4893 not only sterically blocks binding of AT to its cell receptor but also prevents it from adopting a lytic heptameric trans-membrane conformation.

Crystal Structure of the “PhoU-Like” Phosphate Uptake Regulator from Aquifex aeolicus

The phoU gene of Aquifex aeolicus encodes a protein called PHOU_AQUAE with sequence similarity to the PhoU protein of Escherichia coli. Despite the fact that there is a large number of family members (more than 300) attributed to almost all known bacteria and despite PHOU_AQUAEs association with the regulation of genes for phosphate metabolism, the nature of its regulatory function is not well understood. Nearly one-half of these PhoU-like proteins, including both PHOU_AQUAE and the one from E. coli, form a subfamily with an apparent dimer structure of two PhoU domains on the basis of their amino acid sequence. The crystal structure of PHOU_AQUAE (a 221-amino-acid protein) reveals two similar coiled-coil PhoU domains, each forming a three-helix bundle. The structures of PHOU_AQUAE proteins from both a soluble fraction and refolded inclusion bodies (at resolutions of 2.8 and 3.2A, respectively) showed no significant differences. The folds of the PhoU domain and Bag domains (for a class of cofactors of the eukaryotic chaperone Hsp70 family) are similar. Accordingly, we propose that gene regulation by PhoU may occur by association of PHOU_AQUAE with the ATPase domain of the histidine kinase PhoR, promoting release of its substrate PhoB. Other proteins that share the PhoU domain fold include the coiled-coil domains of the STAT protein, the ribosome-recycling factor, and structural proteins like spectrin.

Structure of a NAD kinase from Thermotoga maritima at 2.3 A resolution.

NAD kinase is the only known enzyme that catalyzes the formation of NADP, a coenzyme involved in most anabolic reactions and in the antioxidant defense system. Despite its importance, very little is known regarding the mechanism of catalysis and only recently have several NAD kinase structures been deposited in the PDB. Here, an independent investigation of the crystal structure of inorganic polyphosphate/ATP-NAD kinase, PPNK_THEMA, a protein from Thermotoga maritima, is reported at a resolution of 2.3 A. The crystal structure was solved using single-wavelength anomalous diffraction (SAD) data collected at the Se absorption-peak wavelength in a state in which no cofactors or substrates were bound. It revealed that the 258-amino-acid protein is folded into two distinct domains, similar to recently reported NAD kinases. The N-terminal alpha/beta-domain spans the first 100 amino acids and the last 30 amino acids of the polypeptide and has several topological matches in the PDB, whereas the other domain, which spans the middle 130 residues, adopts a unique beta-sandwich architecture and only appreciably matches the recently deposited PDB structures of NAD kinases.

Structure of O67745_AQUAE, a hypothetical protein from Aquifex aeolicus.

Using single-wavelength anomalous dispersion data obtained from a gold-derivatized crystal, the X-ray crystal structure of the protein 067745_AQUAE from the prokaryotic organism Aquifex aeolicus has been determined to a resolution of 2.0 A. Amino-acid residues 1-371 of the 44 kDa protein were identified by Pfam as an HD domain and a member of the metal-dependent phosphohydrolase superfamily (accession No. PF01966). Although three families from this large and diverse group of enzymatic proteins are represented in the PDB, the structure of 067745_AQUAE reveals a unique fold that is unlike the others and that is likely to represent a new subfamily, further organizing the families and characterizing the proteins. Data are presented that provide the first insights into the structural organization of the proteins within this clan and a distal alternative GDP-binding domain outside the metal-binding active site is proposed.

Structure of the putative DNA-binding protein SP_1288 from Streptococcus pyogenes.

The crystal structure of the putative DNA-binding protein SP_1288 (gi/15675166, also listed as gi/28895954) from Streptococcus pyogenes has been determined by X-ray crystallography to a resolution of 2.3 A using anomalous diffraction data at the Se peak wavelength. SP_1288 belongs to a family of proteins whose cellular function is associated with the signal recognition particle; no structural information has been available until now about the members of the family. Crystallographic analysis revealed that the overall fold of SP_1288 consists exclusively of alpha-helices and that 75% of the structure has good similarity to domain 4 of the sigma subunit of RNA polymerase. This suggests its possible involvement in the biochemical function of transcription initiation, which includes interaction with DNA.