Michael J. Miley

Structural and Functional Consequences of Altering a Peptide MHC Anchor Residue

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.

Evidence for MR1 Antigen Presentation to Mucosal-associated Invariant T Cells

The novel class Ib molecule MR1 is highly conserved in mammals, particularly in its α1/α2 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) α 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/αβTCR 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.

Biochemical Features of the MHC-Related Protein 1 Consistent with an Immunological Function

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.

Structural Basis for the Restoration of TCR Recognition of an MHC Allelic Variant by Peptide Secondary Anchor Substitution

Major histocompatibility complex (MHC) class I variants H-2Kb and H-2Kbm8 differ primarily in the B pocket of the peptide-binding groove, which serves to sequester the P2 secondary anchor residue. This polymorphism determines resistance to lethal herpes simplex virus (HSV-1) infection by modulating T cell responses to the immunodominant glycoprotein B498-505 epitope, HSV8. We studied the molecular basis of these effects and confirmed that T cell receptors raised against Kb–HSV8 cannot recognize H-2Kbm8–HSV8. However, substitution of SerP2 to GluP2 (peptide H2E) reversed T cell receptor (TCR) recognition; H-2Kbm8–H2E was recognized whereas H-2Kb–H2E was not. Insight into the structural basis of this discrimination was obtained by determining the crystal structures of all four MHC class I molecules in complex with bound peptide (pMHCs). Surprisingly, we find no concerted pMHC surface differences that can explain the differential TCR recognition. However, a correlation is apparent between the recognition data and the underlying peptide-binding groove chemistry of the B pocket, revealing that secondary anchor residues can profoundly affect TCR engagement through mechanisms distinct from the alteration of the resting state conformation of the pMHC surface.

Structural Determinants of Affinity Enhancement between GoLoco Motifs and G-Protein α Subunit Mutants

GoLoco motif proteins bind to the inhibitory Gi subclass of G-protein α subunits and slow the release of bound GDP; this interaction is considered critical to asymmetric cell division and neuro-epithelium and epithelial progenitor differentiation. To provide protein tools for interrogating the precise cellular role(s) of GoLoco motif/Gαi complexes, we have employed structure-based protein design strategies to predict gain-of-function mutations that increase GoLoco motif binding affinity. Here, we describe fluorescence polarization and isothermal titration calorimetry measurements showing three predicted Gαi1 point mutations, E116L, Q147L, and E245L; each increases affinity for multiple GoLoco motifs. A component of this affinity enhancement results from a decreased rate of dissociation between the Gα mutants and GoLoco motifs. For Gαi1Q147L, affinity enhancement was seen to be driven by favorable changes in binding enthalpy, despite reduced contributions from binding entropy. The crystal structure of Gαi1Q147L bound to the RGS14 GoLoco motif revealed disorder among three peptide residues surrounding a well defined Leu-147 side chain. Monte Carlo simulations of the peptide in this region showed a sampling of multiple backbone conformations in contrast to the wild-type complex. We conclude that mutation of Glu-147 to leucine creates a hydrophobic surface favorably buried upon GoLoco peptide binding, yet the hydrophobic Leu-147 also promotes flexibility among residues 511–513 of the RGS14 GoLoco peptide.

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

TraI, a bifunctional enzyme containing relaxase and helicase activities, initiates and drives the conjugative transfer of the Escherichia coli F plasmid. Here, we examined the structure and function of the TraI helicase. We show that TraI binds to single-stranded DNA (ssDNA) with a site size of ∼25 nucleotides, which is significantly longer than the site size of other known superfamily I helicases. Low cooperativity was observed with the binding of TraI to ssDNA, and a double-stranded DNA-binding site was identified within the N-terminal region of TraI 1–858, outside the core helicase motifs of TraI. We have revealed that the affinity of TraI for DNA is negatively correlated with the ionic strength of the solution. The binding of AMPPNP or ADP results in a 3-fold increase in the affinity of TraI for ssDNA. Moreover, TraI prefers to bind ssDNA oligomers containing a single type of base. Finally, we elucidated the solution structure of TraI using small angle x-ray scattering. TraI exhibits an ellipsoidal shape in solution with four domains aligning along one axis. Taken together, these data result in the assembly of a model for the multidomain helicase activity of TraI.

Functional Evidence That Conserved TCR CDRα3 Loop Docking Governs the Cross-Recognition of Closely Related Peptide:Class I Complexes

The TCR recognizes its peptide:MHC (pMHC) ligand by assuming a diagonal orientation relative to the MHC helices, but it is unclear whether and to what degree individual TCRs exhibit docking variations when contacting similar pMHC complexes. We analyzed monospecific and cross-reactive recognition by diverse TCRs of an immunodominant HVH-1 glycoprotein B epitope (HSV-8p) bound to two closely related MHC class I molecules, H-2Kb and H-2Kbm8. Previous studies indicated that the pMHC portion likely to vary in conformation between the two complexes resided at the N-terminal part of the complex, adjacent to peptide residues 2–4 and the neighboring MHC side chains. We found that CTL clones sharing TCR β-chains exhibited disparate recognition patterns, whereas those with drastically different TCRβ-chains but sharing identical TCRα CDR3 loops displayed identical functional specificity. This suggested that the CDRα3 loop determines the TCR specificity in our model, the conclusion supported by modeling of the TCR over the actual HSV-8:Kb crystal structure. Importantly, these results indicate a remarkable conservation in CDRα3 positioning, and, therefore, in docking of diverse TCRαβ heterodimers onto variant peptide:class I complexes, implying a high degree of determinism in thymic selection and T cell activation.