20 research outputs found
Engineering aglycosylated antibody variants with immune effector functions
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2009."February 2008." Cataloged from PDF version of thesis.Includes bibliographical references (p. 110-114).Monoclonal antibodies have emerged as a promising class of therapeutics for the treatment of human disease, and in particular human cancer. While multiple mechanisms contribute to antibody efficacy, the engagement and activation of immune effector cells - mediated by the interaction of the conserved Fc regions of the antibody with the Fc gamma receptors (Fc[gamma]Rs) on immune cells - is critical to the efficacy of several. This thesis describes the engineering of antibody Fc domain interactions with Fc[gamma]Rs, using the' yeast S. cerevisiae. In an initial step, a microbial system for the production of full-length antibodies in S. cerevisiae in milligram per liter titers has been developed, which serves as a platform for the engineering of antibody Fc domains with defined properties. The presence of a single N-linked glycan on each chain of the antibody Fc, as well as the specific composition of the glycoforms comprising it, are critical to the binding of the Fc to Fc[gamma]Rs, and have largely limited the production of therapeutic antibodies to mammalian expression systems. Using a display system that tethers full-length antibodies on the surface of yeast, we identify and characterize aglycosylated antibody variants that bind a subset of the human low-affinity Fc[gamma]Rs, Fc[gamma]RIIA and Fc'yRIIB, with approximately wildtype binding affinity and activate immune effector functions in vivo. In a separate approach, we identify aglycosylated variants that weakly bind a third low-affinity receptor, Fc[gamma]RIIIA, and through subsequent engineering generate variants that bind all of the low-affinity Fc[gamma]Rs with approximately wild-type binding affinity. By decoupling the function of the antibody from its post-translational processing, these variants have the potential to open up therapeutic antibody production to a far wider array of expression systems than currently available. Finally, in parallel work, we use a similar system to screen for glycosylated Fc variants with improved affinity and specificity for the activating receptor Fc[gamma]RIIIA compared to the inhibitory receptor Fc[gamma]RIIB, properties which have been hypothesized to lead to more potent antibody therapeutics.by Stephen L. Sazinsky.Ph.D
Component Interactions and Electron Transfer in Toluene/o-Xylene Monooxygenase
The multicomponent protein toluene/o-xylene monooxygenase (ToMO) activates molecular oxygen to oxidize aromatic hydrocarbons. Prior to dioxygen activation, two electrons are injected into each of two diiron(III) units of the hydroxylase, a process that involves three redox active proteins: the ToMO hydroxylase (ToMOH), Rieske protein (ToMOC), and an NADH oxidoreductase (ToMOF). In addition to these three proteins, a small regulatory protein is essential for catalysis (ToMOD). Through steady state and pre-steady state kinetics studies, we show that ToMOD attenuates electron transfer from ToMOC to ToMOH in a concentration-dependent manner. At substoichiometric concentrations, ToMOD increases the rate of turnover, which we interpret to be a consequence of opening a pathway for oxygen transport to the catalytic diiron center in ToMOH. Excess ToMOD inhibits steady state catalysis in a manner that depends on ToMOC concentration. Through rapid kinetic assays, we demonstrate that ToMOD attenuates formation of the ToMOC–ToMOH complex. These data, coupled with protein docking studies, support a competitive model in which ToMOD and ToMOC compete for the same binding site on the hydroxylase. These results are discussed in the context of other studies of additional proteins in the superfamily of bacterial multicomponent monooxygenases.National Institute of General Medical Sciences (U.S.) (5-R01-GM032134)United States. National Institutes of Health (T32GM008334
Control of substrate access to the active site in methane monooxygenase
Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH–MMOB (hereafter termed H–B) complex. Here we remedy this deficiency by providing a crystal structure of H–B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α[subscript 2]β[subscript 2] interface of α[subscript 2]β[subscript 2]γ[subscript 2] MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle.National Institute of General Medical Sciences (U.S.) (Grant GM 32114
X-ray Crystal Structures of Manganese(II)-Reconstituted and Native Toluene/o-Xylene Monooxygenase Hydroxylase Reveal Rotamer Shifts in Conserved Residues and an Enhanced View of the Protein Interior
Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fc Gamma RIIA and Fc Gamma RIIIAs
© 2017 Elsevier Ltd The binding of human IgG1 to human Fc gamma receptors (hFcγRs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs N297D/S298T (DTT)–K326I/A327Y/L328G (IYG) and N297D/S298A–IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT–IYG to be the best at restoring wild-type-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT–IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus, in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function
Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-κB Essential Modulator/IKKβ Protein–Protein Interface
We
report a comprehensive analysis of binding energy hot spots
at the protein–protein interaction (PPI) interface between
nuclear factor kappa B (NF-κB) essential modulator (NEMO) and
IκB kinase subunit β (IKKβ), an interaction that
is critical for NF-κB pathway signaling, using experimental
alanine scanning mutagenesis and also the FTMap method for computational
fragment screening. The experimental results confirm that the previously
identified NEMO binding domain (NBD) region of IKKβ contains
the highest concentration of hot-spot residues, the strongest of which
are W739, W741, and L742 (ΔΔ<i>G</i> = 4.3,
3.5, and 3.2 kcal/mol, respectively). The region occupied by these
residues defines a potentially druggable binding site on NEMO that
extends for ∼16 Å to additionally include the regions
that bind IKKβ L737 and F734. NBD residues D738 and S740 are
also important for binding but do not make direct contact with NEMO,
instead likely acting to stabilize the active conformation of surrounding
residues. We additionally found two previously unknown hot-spot regions
centered on IKKβ residues L708/V709 and L719/I723. The computational
approach successfully identified all three hot-spot regions on IKKβ.
Moreover, the method was able to accurately quantify the energetic
importance of all hot-spot residues involving direct contact with
NEMO. Our results provide new information to guide the discovery of
small-molecule inhibitors that target the NEMO/IKKβ interaction.
They additionally clarify the structural and energetic complementarity
between “pocket-forming” and “pocket-occupying”
hot-spot residues, and further validate computational fragment mapping
as a method for identifying hot spots at PPI interfaces
A specificity map for the PDZ domain family.
PDZ domains are protein-protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position -2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth
Insights into the different dioxygen activation pathways of methane and toluene monooxygenase hydroxylases
The methane and toluene monooxygenase hydroxylases (MMOH and TMOH, respectively) have almost identical active sites, yet the physical and chemical properties of their oxygenated intermediates, designated P*, H[subscript peroxo], Q, and Q* in MMOH and ToMOH[subscript peroxo] in a subclass of TMOH, ToMOH, are substantially different. We review and compare the structural differences in the vicinity of the active sites of these enzymes and discuss which changes could give rise to the different behavior of H[subscript peroxo] and Q. In particular, analysis of multiple crystal structures reveals that T213 in MMOH and the analogous T201 in TMOH, located in the immediate vicinity of the active site, have different rotatory configurations. We study the rotational energy profiles of these threonine residues with the use of molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) computational methods and put forward a hypothesis according to which T213 and T201 play an important role in the formation of different types of peroxodiiron(III) species in MMOH and ToMOH. The hypothesis is indirectly supported by the QM/MM calculations of the peroxodiiron(III) models of ToMOH and the theoretically computed Mössbauer spectra. It also helps explain the formation of two distinct peroxodiiron(III) species in the T201S mutant of ToMOH. Additionally, a role for the ToMOD regulatory protein, which is essential for intermediate formation and protein functioning in the ToMO system, is advanced. We find that the low quadrupole splitting parameter in the Mössbauer spectrum observed for a ToMOHperoxo intermediate can be explained by protonation of the peroxo moiety, possibly stabilized by the T201 residue. Finally, similarities between the oxygen activation mechanisms of the monooxygenases and cytochrome P450 are discussed.National Institute of General Medical Sciences (U.S.) (grant no.GM032134