N-RAS mutation at codon 12, 13 or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an N-RasQ61R knock-in allele to similarly designed K-RasG12D and N-RasG12D alleles. With concomitant p16INK4a inactivation, K-RasG12D or N-RasQ61R expression efficiently promoted melanoma in vivo, whereas N-RasG12D did not. Additionally, N-RasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of N-RasQ61R and N-RasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, N-RasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity and increased stability when compared to N-RasG12D. This work identifies a faithful model of human N-RAS mutant melanoma, and suggests that the increased melanomagenecity of N-RasQ61R over N-RasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways.
The novel class Ib molecule MR1 is highly conserved in mammals, particularly in its ␣1/␣2 domains. Recent studies demonstrated that MR1 expression is required for development and expansion of a small population of T cells expressing an invariant T cell receptor (TCR) ␣ chain called mucosal-associated invariant T (MAIT) cells. Despite these intriguing properties it has been difficult to determine whether MR1 expression and MAIT cell recognition is ligand-dependent. To address these outstanding questions, monoclonal antibodies were produced in MR1 knock-out mice immunized with recombinant MR1 protein, and a series of MR1 mutations were generated at sites previously shown to disrupt the ability of class Ia molecules to bind peptide or TCR. Here we show that 1) MR1 molecules are detected by monoclonal antibodies in either an open or folded conformation that correlates precisely with peptide-induced conformational changes in class Ia molecules, 2) only the folded MR1 conformer activated 2/2 MAIT hybridoma cells tested, 3) the pattern of MAIT cell activation by the MR1 mutants implies the MR1/TCR orientation is strikingly similar to published major histocompatibility complex/␣TCR engagements, 4) all the MR1 mutations tested and found to severely reduce surface expression of folded molecules were located in the putative ligand binding groove, and 5) certain groove mutants of MR1 that are highly expressed on the cell surface disrupt MAIT cell activation. These combined data strongly support the conclusion that MR1 has an antigen presentation function.
SummaryHuman UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in the processing of a range of endobiotic compounds. UGTs catalyze the covalent addition of glucuronic acid sugar moieties to a host of therapeutics and environmental toxins, as well as to a variety of endogenous steroids and other signaling molecules. We report the 1.8 Å resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT isoform 2B7 (UGT2B7), which catalyzes the conjugative elimination of opioid, antiviral, and anticancer drugs. This is the first crystal structure of any region of a mammalian UGT drug metabolism enzyme. Designed UGT2B7 mutants at residues predicted to interact with the UDP-glucuronic acid cofactor exhibited significantly impaired catalytic activity, with maximum effects observed for amino acids closest to the glucuronic acid sugar transferred to the acceptor molecule. Homology modeling of UGT2B7 with related plant flavonoid glucosyltransferases indicate that human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting that human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer.
MHC-related protein (MR)1 is an MHC class I-related molecule encoded on chromosome 1 that is highly conserved among mammals and is more closely related to classical class I molecules than are other nonclassical class I family members. In this report, we show for the first time that both mouse and human MR1 molecules can associate with the peptide-loading complex and can be detected at low levels at the surface of transfected cells. We also report the production of recombinant human MR1 molecules in insect cells using highly supplemented media and provide evidence that the MR1 H chain can assume a folded conformation and is stoichiometrically associated with β2-microglobulin, similar to class I molecules. Cumulatively, these findings demonstrate that surface expression of MR1 is possible but may be limited by a specific ligand or associated molecule.
Computationally designing protein-protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer. Our metal interface design, named MID1 (NESG target ID OR37), forms a tight dimer in the presence of zinc (MID1-zinc) with a dissociation constant <30 nM. Without zinc the dissociation constant is 4 μM. The crystal structure of MID1-zinc shows good overall agreement with the computational model, but only three out of four designed histidines coordinate zinc. However, a histidine-to-glutamate point mutation resulted in four-coordination of zinc, and the resulting metal binding site and dimer orientation closely matches the computational model (Cα RMSD = 1.4 Å).
Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at sub-nanomolar concentrations. Furthermore, the lead compound shows a promising selectivity profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also describe the crystal structure of the complex between the lead compound and Mer, opening new opportunities for further optimization and new template design.
Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide,  2 -microglobulin, and heavy chain are attached by flexible linkers. These molecules, singlechain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules. In this study, we used a structurebased approach to engineer an F pocket variant SCT of the murine class I molecule K b that presents the SIIN-FEKL epitope of ovalbumin. Mutation of heavy chain residue Tyr 84 (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide. These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide. In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli. SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide⅐class I complexes prone to dissociation. Major histocompatibility complex (MHC)1 class I molecules are heterotrimers consisting of a transmembrane (type I) heavy chain, a soluble light chain ( 2 -microglobulin ( 2 m)), and a peptide ligand of typically 8 -10 amino acids. The initial assembly of the class I heterotrimers occurs in the endoplasmic reticulum (ER) and requires the concerted activity of several accessory molecules. Complete assembly of the heterotrimer is necessary for ER egress and stable surface expression of class I molecules (1, 2). On the cell surface, class I molecules containing the appropriate peptides can mark pathogen-infected or malignant cells for elimination by cytolytic CD8 T lymphocytes (CTL). As clear evidence of the importance of class I antigen presentation, numerous virus-encoded proteins that block various steps in the class I assembly pathway to evade host immune responses have been identified (3).Studies of the structure and function of several different oligomeric immune receptors have been facilitated by the covalent attachment of various subunits via flexible linkers. This is particularly true in the case of MHC class II molecules. The peptide ligand-binding site of MHC class II molecules, in contrast to class I molecules, is formed by the interaction of two separate membrane-bound chains designated ␣ and . To produce MHC class II molecules occupied by homogeneous peptides, constructs were made whereby different peptides were attached to the N terminus of the class II -chain using flexible linkers (4). Class II molecules with covalently bound single peptides have been very informative in studies of class II assembly and importance of chaperone molecules in optimal class II peptide loading (4 -6). Furthermore, such class II molecules with covalently attached peptides have provided insights into CD4...
To better understand TCR discrimination of multiple ligands, we have analyzed the crystal structures of two Hb peptide/I-Ek complexes that differ by only a single amino acid substitution at the P6 anchor position within the peptide (E73D). Detailed comparison of multiple independently determined structures at 1.9 Å resolution reveals that removal of a single buried methylene group can alter a critical portion of the TCR recognition surface. Significant variance was observed in the peptide P5-P8 main chain as well as a rotamer difference at LeuP8, ∼10 Å distal from the substitution. No significant variations were observed in the conformation of the two MHC class II molecules. The ligand alteration results in two peptide/MHC complexes that generate bulk T cell responses that are distinct and essentially nonoverlapping. For the Hb-specific T cell 3.L2, substitution reduces the potency of the ligand 1000-fold. Soluble 3.L2 TCR binds the two peptide/MHC complexes with similar affinity, although with faster kinetics. These results highlight the role of subtle variations in MHC Ag presentation on T cell activation and signaling.
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