Alternative Splice Transcripts for MHC Class I-like MICA Encode Novel NKG2D Ligands with Agonist or Antagonist Functions.

MHC class I chain-related proteins A and B (MICA and MICB) and UL16-binding proteins are ligands of the activating NKG2D receptor involved in cancer and immune surveillance of infection. Structurally, MICA/B proteins contain an α3 domain, whereas UL16-binding proteins do not. We identified novel alternative splice transcripts for MICA encoding five novel MICA isoforms: MICA-A, -B1, -B2, -C, and -D. Alternative splicing associates with MICA*015 and *017 and results from a point deletion (G) in the 5' splice donor site of MICA intron 4 leading to exon 3 and exon 4 skipping and/or deletions. These changes delete the α3 domain in all isoforms, and the α2 domain in the majority of isoforms (A, B1, C, and D). Endothelial and hematopoietic cells contained endogenous alternative splice transcripts and isoforms. MICA-B1, -B2, and -D bound NKG2D by surface plasmon resonance and were expressed at the cell surface. Functionally, MICA-B2 contains two extracellular domains (α1 and α2) and is a novel potent agonist ligand for NKG2D. We found that MICA-D is a new truncated form of MICA with weak affinity for NKG2D despite lacking α2 and α3 domains. MICA-D may functionally impair NKG2D activation by competing with full-length MICA or MICA-B2 for NKG2D engagement. Our study established NKG2D binding for recombinant MICA-B1 but found no function for this isoform. New truncated MICA isoforms exhibit a range of functions that may drive unexpected immune mechanisms and provide new tools for immunotherapy.

Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence.

We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This seven-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies, which are collected, washed, then solubilized in urea. Stepwise dialysis to dilute urea over the course of a week produces refolded protein. Column chromatography is used to purify protein into fractions, which are then analyzed with gel electrophoresis and concentration assays. Students culminate the project by designing crystallization trials in sitting-drop trays. Student evaluation of the experience has been positive, listing 5-12 new techniques learned, which are transferrable to graduate research in academia and industry.

Energetics and cooperativity of the hydrogen bonding and anchor interactions that bind peptides to MHC class II protein.

The complexity of the interaction between major histocompatibility complex class II (MHC II) proteins and peptide ligands has been revealed through structural studies and crystallographic characterization. Peptides bind through side-chain "anchor" interactions with MHC II pockets and an extensive array of genetically conserved hydrogen bonds to the peptide backbone. Here we quantitatively investigate the kinetic hierarchy of these interactions. We present results detailing the impact of single side-chain mutations of peptide anchor residues on dissociation rates, utilizing two I-A(d)-restricted peptides, one of which has a known crystal structure, and 24 natural and non-natural amino acid mutant variants of these peptides. We find that the N-terminal P1, P4 and P6 anchor-pocket interactions can make significant contributions to binding stability. We also investigate the interactions of these peptides with four I-A(d) MHC II proteins, each mutated to disrupt conserved hydrogen bonds to the peptide backbone. These complexes exhibit kinetic behavior suggesting that binding energy is disproportionately invested near the peptide N terminus for backbone hydrogen bonds. We then evaluate the effects of simultaneously modifying both anchor and hydrogen bonding interactions. A quantitative analysis of 71 double mutant cycles reveals that there is little apparent cooperativity between anchor residue interactions and hydrogen bonds, even when they are directly adjacent (<5A).

Symmetry recognizing asymmetry: analysis of the interactions between the C-type lectin-like immunoreceptor NKG2D and MHC class I-like ligands.

Engagement of diverse protein ligands (MIC-A/B, ULBP, Rae-1, or H60) by NKG2D immunoreceptors mediates elimination of tumorigenic or virally infected cells by natural killer and T cells. Three previous NKG2D-ligand complex structures show the homodimeric receptor interacting with the monomeric ligands in similar 2:1 complexes, with an equivalent surface on each NKG2D monomer binding intimately to a total of six distinct ligand surfaces. Here, the crystal structure of free human NKG2D and in silico and in vitro alanine-scanning mutagenesis analyses of the complex interfaces indicate that NKG2D recognition degeneracy is not explained by a classical induced-fit mechanism. Rather, the divergent ligands appear to utilize different strategies to interact with structurally conserved elements of the consensus NKG2D binding site.

NKG2D and Related Immunoreceptors.

NK cells are crucial components of the innate immune system, capable of directly eliminating infected or tumorigenic cells and regulating down-stream adaptive immune responses. Unlike T cells, where the key recognition event driving activation is mediated by the unique T cell receptor (TCR) expressed on a given cell, NK cells express multiple activating and inhibitory cell-surface receptors (NKRs), often with overlapping ligand specificities. NKRs display two ectodomain structural homologies, either immunoglobulin- or C-type lectin-like (CTLD). The CTLD immunoreceptor NKG2D is found on NK cells but is also widely expressed on T cells and other immune system cells, providing stimulatory or co-stimulatory signals. NKG2D drives target cell killing following engagement of diverse, conditionally expressed MHC class I-like protein ligands whose expression can signal cellular distress due to infection or transformation. The symmetric, homodimeric receptor interacts with its asymmetric, monomeric ligands in similar 2:1 complexes, with an equivalent surface on each NKG2D monomer binding extensively and intimately to distinct, structurally divergent surfaces on the ligands. Thus, NKG2D ligand-binding site recognition is highly degenerate, further demonstrated by NKG2D's ability to simultaneously accommodate multiple non-conservative allelic or isoform substitutions in the ligands. In TCRs, "induced-fit" recognition explains cross-reactivity, but structural, computational, thermodynamic and kinetic analyses of multiple NKG2D-ligand pairs show that rather than classical "induced-fit" binding, NKG2D degeneracy is achieved using distinct interaction mechanisms at each rigid interface: recognition degeneracy by "rigid adaptation." While likely forming similar complexes with their ligand (HLA-E), other NKG2x NKR family members do not require such recognition degeneracy.

Combining different design strategies for rational affinity maturation of the MICA-NKG2D interface.

We redesigned residues on the surface of MICA, a protein that binds the homodimeric immunoreceptor NKG2D, to increase binding affinity with a series of rational, incremental changes. A fixed-backbone RosettaDesign protocol scored a set of initial mutations, which we tested by surface plasmon resonance for thermodynamics and kinetics of NKG2D binding, both singly and in combination. We combined the best four mutations at the surface with three affinity-enhancing mutations below the binding interface found with a previous design strategy. After curating design scores with three cross-validated tests, we found a linear relationship between free energy of binding and design score, and to a lesser extent, enthalpy and design score. Multiple mutants bound with substantial subadditivity, but in at least one case full additivity was observed when combining distant mutations. Altogether, combining the best mutations from the two strategies into a septuple mutant enhanced affinity by 50-fold, to 50 nM, demonstrating a simple, effective protocol for affinity enhancement.

Systematic mutation and thermodynamic analysis of central tyrosine pairs in polyspecific NKG2D receptor interactions.

The homodimeric, activating natural killer cell receptor NKG2D interacts with multiple monomeric ligands polyspecifically, yet without central conformational flexibility. Crystal structures of multiple NKG2D-ligand interactions have identified the NKG2D tyrosine pair Tyr 152 and Tyr 199 as forming multiple specific but diverse interactions with MICA and related proteins. Here we systematically altered each tyrosine to tryptophan, phenylalanine, isoleucine, leucine, valine, serine, and alanine to measure the effect of mutation on affinity and thermodynamics for binding a range of similar ligands: MICA, the higher-affinity ligand MICB, and MICdesign, a high-affinity version of MICA that shares all NKG2D contact residues with MICA. Affinity and residue size were related: tryptophan could often substitute for tyrosine without loss of affinity; loss of the tyrosine hydroxyl through mutation to phenylalanine was tolerated more at position 152 than 199; and the smallest residues coincide with lowest affinities in general. NKG2D mutant van't Hoff binding thermodynamics generally show that substitution of other residues for tyrosine causes a moderate positive or flat van't Hoff slope consistent with moderate loss of binding enthalpy. One set of NKG2D mutations caused MICA to adopt a positive van't Hoff slope corresponding to absorption of heat, and another set caused MICB to adopt a negative slope of greater heat release than wild-type. MICdesign shared one example of the first set with MICA and one of the second set with MICB. When the NKG2D mutation affinities were arranged according to change in nonpolar surface area and compared to results from specific antibody-antigen and protein-peptide interactions, it was found that hydrophobic surface loss in NKG2D reduced binding affinity less than reported in the other contexts. The hydrophobic effect at the center of the NKG2D binding appears more similar to that at the periphery of an antibody-antigen binding site than at its center. Therefore the polyspecific NKG2D binding site is more tolerant of structural alteration in general than either an antibody-antigen or protein-peptide binding site, and this tolerance may adapt NKG2D to a broad range of protein surfaces with micromolar affinity.

Size-exclusion chromatography can identify faster-associating protein complexes and evaluate design strategies.

BACKGROUND: We previously developed a set of rationally designed mutant MICA protein ligands for the NKG2D immunoreceptor in which MICA was mutated at residues that do not contact NKG2D. Some of these MICA mutants, predicted by RosettaDesign to be destabilized, bound NKG2D with affinities enhanced by more than an order of magnitude when evaluated by surface plasmon resonance (SPR). FINDINGS: Small-zone size-exclusion chromatography (SEC) detected persistent high-affinity MICA mutant-NKG2D complexes in solution as early-eluting peaks. The SEC binding assay used standard protein purification instrumentation to evaluate complex stability, qualitatively paralleled the SPR results, and successfully discriminated among complexes that differed only in on-rates. We used the SEC binding assay, along with SPR, to assess the results of a follow-up design strategy targeting the non-interfacial redesigned region. Both SEC and SPR agreed that these mutations did not enhance affinity as much as previous mutants. When the SEC binding assay was run in 1 M urea, only the highest affinity complex was detected. CONCLUSION: This SEC binding assay provides a correlation with SPR results for protein complex affinities, detecting changes in complex on-rates, and tunable to lower sensitivity with 1 M urea. The SEC binding assay is complementary to other protein design evaluation methods, can be adapted to the undergraduate research laboratory, and may provide additional structural information about changes in hydrodynamic radii from elution times. Our assay allowed us to conclude that further alteration of MICA at non-contacting residues is unlikely to further enhance NKG2D affinity.

Mutations designed to destabilize the receptor-bound conformation increase MICA-NKG2D association rate and affinity.

MICA is a major histocompatibility complex-like protein that undergoes a structural transition from disorder to order upon binding its immunoreceptor, NKG2D. We redesigned the disordered region of MICA with RosettaDesign to increase NKG2D binding. Mutations that stabilize this region were expected to increase association kinetics without changing dissociation kinetics, increase affinity of interaction, and reduce entropy loss upon binding. MICA mutants were stable in solution, and they were amenable to surface plasmon resonance evaluation of NKG2D binding kinetics and thermodynamics. Several MICA mutants bound NKG2D with enhanced affinity, kinetic changes were primarily observed during association, and thermodynamic changes in entropy were as expected. However, none of the 15 combinations of mutations predicted to stabilize the receptor-bound MICA conformation enhanced NKG2D affinity, whereas all 10 mutants predicted to be destabilized bound NKG2D with increased on-rates. Five of these had affinities enhanced by 0.9-1.8 kcal/mol over wild type by one to three non-contacting substitutions. Therefore, in this case, mutations designed to mildly destabilize a protein enhanced association and affinity.

Binding interactions between peptides and proteins of the class II major histocompatibility complex.

The activation of helper T cells by peptides bound to proteins of the class II Major Histocompatibility Complex (MHC II) is pivotal to the initiation of an immune response. The primary functional requirement imposed on MHC II proteins is the ability to efficiently bind thousands of different peptides. Structurally, this is reflected in a unique architecture of binding interactions. The peptide is bound in an extended conformation within a groove on the membrane distal surface of the protein that is lined with several pockets that can accommodate peptide side-chains. Conserved MHC II protein residues also form hydrogen bonds along the length of the peptide main-chain. Here we review recent advances in the study of peptide-MHC II protein reactions that have led to an enhanced understanding of binding energetics. These results demonstrate that peptide-MHC II protein complexes achieve high affinity binding from the array of hydrogen bonds that are energetically segregated from the pocket interactions, which can then add to an intrinsic hydrogen bond-mediated affinity. Thus, MHC II proteins are unlike antibodies, which utilize cooperativity among binding interactions to achieve high affinity and specificity. The significance of these observations is discussed within the context of possible mechanisms for the HLA-DM protein that regulates peptide presentation in vivo and the design of non-peptide molecules that can bind MHC II proteins and act as vaccines or immune modulators.

Thermodynamic analysis of degenerate recognition by the NKG2D immunoreceptor: not induced fit but rigid adaptation.

The homodimeric immunoreceptor NKG2D drives the activation of effector cells following engagement of diverse, conditionally expressed MHC class I-like protein ligands. NKG2D recognition is highly degenerate in that a single surface on receptor monomers binds pairs of distinct surfaces on each structurally divergent ligand, simultaneously accommodating multiple nonconservative ligand allelic or isoform substitutions. In contrast to TCR-pMHC and other NK receptor-ligand interactions, thermodynamic and kinetic analyses of four NKG2D-ligand pairs (MIC-A*001, MIC-B*005, ULBP1, and RAE-1beta) reported here show that the relative enthalpic and entropic terms, heat capacity, association rates, and activation energy barriers are comparable to typical, rigid protein-protein interactions. Rather than "induced-fit" binding, NKG2D degeneracy is achieved using distinct interaction mechanisms at each rigid interface.