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A challenge in the structure-based design of specificity is modeling the negative states, i.e., the complexes that you do not want to form. This is a difficult problem because mutations predicted to destabilize the negative state might be accommodated by small conformational rearrangements. To overcome this challenge, we employ an iterative strategy that cycles between sequence design and protein docking in order to build up an ensemble of alternative negative state conformations for use in specificity prediction. We have applied our technique to the design of heterodimeric CH3 interfaces in the Fc region of antibodies. Combining computationally and rationally designed mutations produced unique designs with heterodimer purities greater than 90%. Asymmetric Fc crystallization was able to resolve the interface mutations; the heterodimer structures confirmed that the interfaces formed as designed. With these CH3 mutations, and those made at the heavy-/light-chain interface, we demonstrate one-step synthesis of four fully IgG-bispecific antibodies.

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Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain-light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

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Improved affinity for the neonatal Fc receptor (FcRn) is known to extend antibody half-life in vivo. However, this has never been linked with enhanced therapeutic efficacy. We tested whether antibodies with half-lives extended up to fivefold in human (h)FcRn transgenic mice and threefold in cynomolgus monkeys retain efficacy at longer dosing intervals. We observed that prolonged exposure due to FcRn-mediated enhancement of half-life improved antitumor activity of Fc-engineered antibodies in an hFcRn/Rag1(-/-) mouse model. This bridges the demand for dosing convenience with the clinical necessity of maintaining efficacy.

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We measured the frequency of side-chain rotamers in 14 alpha-helical and 16 beta-barrel membrane protein structures and found that the membrane environment considerably perturbs the rotamer frequencies compared to soluble proteins. Although there are limited experimental data, we found statistically significant changes in rotamer preferences depending on the residue environment. Rotamer distributions were influenced by whether the residues were lipid or protein facing, and whether the residues were found near the N- or C-terminus. Hydrogen-bonding interactions with the helical backbone perturbs the rotamer populations of Ser and His. Trp and Tyr favor side-chain conformations that allow their side chains to extend their polar atoms out of the membrane core, thereby aligning the side-chain polarity gradient with the polarity gradient of the membrane. Our results demonstrate how the membrane environment influences protein structures, providing information that will be useful in the structure prediction and design of transmembrane proteins.

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The nicotinic acetylcholine receptor is a neurotransmitter-gated ion channel in the postsynaptic membrane. It is composed of five homologous subunits, each of which contributes one transmembrane helix--the M2 helix--to create the channel pore. The M2 helix from the delta subunit is capable of forming a channel by itself. Although a model of the receptor was recently proposed based on a low-resolution, cryo-electron microscopy density map, we found that the model does not explain much of the other available experimental data. Here we propose a new model of the M2 channel derived solely from helix packing and symmetry constraints. This model agrees well with experimental results from solid-state NMR, chemical reactivity, and mutagenesis experiments. The model depicts the channel pore, the channel gate, and the residues responsible for cation specificity.

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In contrast to water-soluble proteins, membrane proteins reside in a heterogeneous environment, and their surfaces must interact with both polar and apolar membrane regions. As a consequence, the composition of membrane proteins' residues varies substantially between the membrane core and the interfacial regions. The amino acid compositions of helical membrane proteins are also known to be different on the cytoplasmic and extracellular sides of the membrane. Here we report that in the 16 transmembrane beta-barrel structures, the amino acid compositions of lipid-facing residues are different near the N and C termini of the individual strands. Polar amino acids are more prevalent near the C termini than near the N termini, and hydrophobic amino acids show the opposite trend. We suggest that this difference arises because it is easier for polar atoms to escape from the apolar regions of the bilayer at the C terminus of a beta-strand. This new characteristic of beta-barrel membrane proteins enhances our understanding of how a sequence encodes a membrane protein structure and should prove useful in identifying and predicting the structures of trans-membrane beta-barrels.

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By analyzing transmembrane (TM) helices in known structures, we find that some polar amino acids are more frequent at the N terminus than at the C terminus. We propose the asymmetry occurs because most polar amino acids are better able to snorkel their polar atoms away from the membrane core at the N terminus than at the C terminus. Two findings lead us to this proposition: (1) side-chain conformations are influenced strongly by the N or C-terminal position of the amino acid in the bilayer, and (2) the favored snorkeling direction of an amino acid correlates well with its N to C-terminal composition bias. Our results suggest that TM helix predictions should incorporate an N to C-terminal composition bias, that rotamer preferences of TM side-chains are position-dependent, and that the ability to snorkel influences the evolutionary selection of amino acids for the helix N and C termini.

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Helicobacter pylori is a human pathogen responsible for severe gastric diseases such as peptic ulcers, gastric adenocarcinoma, and gastric lymphoma. Vacuolating toxin (VacA) is crucial in facilitating the colonization of the gastric lining by inducing cell apoptosis and immune suppression. VacA inserts into membranes and forms a hexameric, anion-selective pore. Here we present a structural model of the VacA pore that strongly resembles the structure of an unrelated anion-selective channel, MscS. In our model, Gly residues in GXXXG motifs pack against small Ala or Val side chains to generate the pore. Our model suggests that the same design of two anion-selective channels was achieved by two different evolutionary paths and provides insight into the mechanism of VacA function.

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Hydrogen bonds involving a carbon donor are very common in protein structures, and energy calculations suggest that Calpha-H...O hydrogen bonds could be about one-half the strength of traditional hydrogen bonds. It has therefore been proposed that these nontraditional hydrogen bonds could be a significant factor in stabilizing proteins, particularly membrane proteins as there is a low dielectric and no competition from water in the bilayer core. Nevertheless, this proposition has never been tested experimentally. Here, we report an experimental test of the significance of Calpha-H...O bonds for protein stability. Thr24 in bacteriorhodopsin, which makes an interhelical Calpha-H...O hydrogen bond to the Calpha of Ala51, was changed to Ala, Val, and Ser, and the thermodynamic stability of the mutants was measured. None of the mutants had significantly reduced stability. In fact, T24A was more stable than the wild-type protein by 0.6 kcal/mol. Crystal structures were determined for each of the mutants, and, while some structural changes were seen for T24S and T24V, T24A showed essentially no apparent structural alteration that could account for the increased stability. Thus, Thr24 appears to destabilize the protein rather than stabilize. Our results suggest that Calpha-H...O bonds are not a major contributor to protein stability.

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We describe an effective procedure for modeling the structures of simple transmembrane helix homo-oligomers. The method differs from many previous approaches in that the only structural constraint we use to help select the correct model is the oligomerization state of the protein. The method involves the following steps: (1) perform 100-250 independent Monte Carlo energy minimizations of helix pairs to produce a large collection of well-packed structures; (2) filter the minimized structures to find those that are consistent with the expected symmetry of the oligomer; (3) cluster the structures that pass the symmetry filter; and (4) select a representative of the most populous cluster as the final prediction. We applied the method to the transmembrane helices of five proteins and compare our results to the available experimental data. Our predictions of glycophorin A, neu, the M2 channel and phospholamban resulted in a single model for each protein that agreed with the experimental results. In the case of erbB-2, however, we obtained three structurally distinct clusters of approximately equal sizes, so it was not possible to identify a clearly favored structure. This may reflect a real heterogeneity of packing modes for erbB-2, which is known to interact with different receptor subunits. Our method should be useful for obtaining structural models of transmembrane domains, improving our understanding of structure/function relationships for particular membrane proteins.

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Non-traditional C-H cdots, three dots, centered Y hydrogen bonds, in which a carbon atom acts as the hydrogen donor and an electronegative atom Y (Y=N, O or S) acts as the acceptor, have been reported in proteins, but their importance in protein structures is not well established. Here, we present the results of three computational tests that examine the significance of C-H cdots, three dots, centered Y bonds involving the C(alpha) in proteins. First, we compared the number of C(alpha)-H cdots, three dots, centered Y bonds in native structures with two sets of compact, energy-minimized decoy structures. The decoy structures contain about as many C(alpha)-H cdots, three dots, centered Y bonds as the native structures, indicating that the constraints of chain connectivity and compactness can lead to incidental formation of C(alpha)-H cdots, three dots, centered Y bonds. Secondly, we examined whether short C(alpha)-H cdots, three dots, centered Y bonds have a tendency to be linear, as is expected for a cohesive hydrogen-bonding interaction. The native structures do show this trend, but so does one of the decoy sets, suggesting that this criterion is also not sufficient to indicate a stabilizing interaction. Finally, we examined the preference for C(alpha)-H cdots, three dots, centered Y bond donors to be near to strong hydrogen bond acceptors. In the native proteins, the alpha protons attract strong acceptors like oxygen atoms more than weak acceptors. In contrast, hydrogen bond donors in the decoy structures do not distinguish between strong and weak acceptors. Thus, any individual C(alpha)-H cdots, three dots, centered Y bond may be fortuitous and occur due to the polypeptide connectivity and compactness. Taken collectively, however, C(alpha)-H cdots, three dots, centered Y bonds provide a weakly cohesive force that stabilizes proteins.

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