A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent Ig-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full-length and truncated forms of the MHC-I molecule H2-L^d by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-L^d, “mini-H2-L^d,” consisting of only the α1α2 platform domain. Mini-H2-L^d refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the β₂-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and β₂-microglobulin-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the picosecond-nanosecond dynamics of the free mini-H2-L^d MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.     

Solution structure of the strawberry allergen Fra a 1

The PR10 family protein Fra a 1E from strawberry (Fragaria x ananassa) is down-regulated in white strawberry mutants, and transient RNAi (RNA interference)-mediated silencing experiments confirmed that Fra a 1 is involved in fruit pigment synthesis. In the present study, we determined the solution structure of Fra a 1E. The protein fold is identical with that of other members of the PR10 protein family and consists of a seven-stranded antiparallel β-sheet, two short V-shaped α-helices and a long C-terminal α-helix that encompass a hydrophobic pocket. Whereas Fra a 1E contains the glycine-rich loop that is highly conserved throughout the protein family, the volume of the hydrophobic pocket and the size of its entrance are much larger than expected. The three-dimensional structure may shed some light on its physiological function and may help to further understand the role of PR10 proteins in plants.     

Small Molecule Agonist of Very Late Antigen-4 (VLA-4) Integrin Induces Progenitor Cell Adhesion

Activation of the integrin family of cell adhesion receptors on progenitor cells may be a viable approach to enhance the effects of stem cell-based therapies by improving cell retention and engraftment. Here, we describe the synthesis and characterization of the first small molecule agonist identified for the integrin α4β1 (also known as very late antigen-4 or VLA-4). The agonist, THI0019, was generated via two structural modifications to a previously identified α4β1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to α4β1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1α. Molecular modeling predicted that the compound binds at the α/β subunit interface overlapping the ligand-binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin α4β7 as well as α5β1 and αLβ2. When cross-linked to αLβ2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the α/β subunit interface and not at the ligand-binding site in the inserted (“I”) domain of the αL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.     

Crystal structure of the murine cytomegalovirus MHC-I homolog m144

Large DNA viruses of the herpesvirus family produce proteins that mimic host MHC-I molecules as part of their immunoevasive strategy. The m144 glycoprotein, expressed by murine cytomegalovirus, is thought to be an MHC-I homolog whose expression prolongs viral survival in vivo by preventing natural killer cell activation. To explore the structural basis of this m144 function, we have determined the three-dimensional structure of an m144/beta2-microglobulin (beta2m) complex at 1.9A resolution. This structure reveals the canonical features of MHC-I molecules including readily identifiable alpha1, alpha2, and alpha3 domains. A unique disulfide bond links the alpha1 helix to the beta-sheet floor, explaining the known thermal stability of m144. Close juxtaposition of the alpha1 and alpha2 helices and the lack of critical residues that normally contribute to anchoring the peptide N and C termini eliminates peptide binding. A region of 13 amino acid residues, corresponding to the amino-terminal portion of the alpha2 helix, is missing in the electron density map, suggesting an area of structural flexibility that may be involved in ligand binding.     

Antigen-derived peptides engage the ER stress sensor IRE1α to curb dendritic cell cross-presentation

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8⁺ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER), bind to major histocompatibility type I (MHC-I) protein complexes, and are transported to the cell surface for cross-presentation. DCs can exhibit activation of the ER stress sensor IRE1α without ER stress, but the underlying mechanism remains obscure. Here, we show that antigen-derived hydrophobic peptides can directly engage ER-resident IRE1α, masquerading as unfolded proteins. IRE1α activation depletes MHC-I heavy-chain mRNAs through regulated IRE1α-dependent decay (RIDD), curtailing antigen cross-presentation. In tumor-bearing mice, IRE1α disruption increased MHC-I expression on tumor-infiltrating DCs and enhanced recruitment and activation of CD8⁺ T cells. Moreover, IRE1α inhibition synergized with anti–PD-L1 antibody treatment to cause tumor regression. Our findings identify an unexpected cell-biological mechanism of antigen-driven IRE1α activation in DCs, revealing translational potential for cancer immunotherapy.     

Aromatic cluster mutations produce focal modulations of β-sheet structure

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain “clusters” at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design.     

Organization of the Mitochondrial Apoptotic BAK Pore: OLIGOMERIZATION OF THE BAK HOMODIMERS*

The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX “BH3-in-groove homodimer.” Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a “worm hole.”     

Structural Evolution of the Protein Kinase–Like Superfamily

The protein kinase family is large and important, but it is only one family in a larger superfamily of homologous kinases that phosphorylate a variety of substrates and play important roles in all three superkingdoms of life. We used a carefully constructed structural alignment of selected kinases as the basis for a study of the structural evolution of the protein kinase–like superfamily. The comparison of structures revealed a “universal core” domain consisting only of regions required for ATP binding and the phosphotransfer reaction. Remarkably, even within the universal core some kinase structures display notable changes, while still retaining essential activity. Hence, the protein kinase–like superfamily has undergone substantial structural and sequence revision over long evolutionary timescales. We constructed a phylogenetic tree for the superfamily using a novel approach that allowed for the combination of sequence and structure information into a unified quantitative analysis. When considered against the backdrop of species distribution and other metrics, our tree provides a compelling scenario for the development of the various kinase families from a shared common ancestor. We propose that most of the so-called “atypical kinases” are not intermittently derived from protein kinases, but rather diverged early in evolution to form a distinct phyletic group. Within the atypical kinases, the aminoglycoside and choline kinase families appear to share the closest relationship. These two families in turn appear to be the most closely related to the protein kinase family. In addition, our analysis suggests that the actin-fragmin kinase, an atypical protein kinase, is more closely related to the phosphoinositide-3 kinase family than to the protein kinase family. The two most divergent families, α-kinases and phosphatidylinositol phosphate kinases (PIPKs), appear to have distinct evolutionary histories. While the PIPKs probably have an evolutionary relationship with the rest of the kinase superfamily, the relationship appears to be very distant (and perhaps indirect). Conversely, the α-kinases appear to be an exception to the scenario of early divergence for the atypical kinases: they apparently arose relatively recently in eukaryotes. We present possible scenarios for the derivation of the α-kinases from an extant kinase fold.     

The Natural History of Biocatalytic Mechanisms

Phylogenomic analysis of the occurrence and abundance of protein domains in proteomes has recently showed that the α/β architecture is probably the oldest fold design. This holds important implications for the origins of biochemistry. Here we explore structure-function relationships addressing the use of chemical mechanisms by ancestral enzymes. We test the hypothesis that the oldest folds used the most mechanisms. We start by tracing biocatalytic mechanisms operating in metabolic enzymes along a phylogenetic timeline of the first appearance of homologous superfamilies of protein domain structures from CATH. A total of 335 enzyme reactions were retrieved from MACiE and were mapped over fold age. We define a mechanistic step type as one of the 51 mechanistic annotations given in MACiE, and each step of each of the 335 mechanisms was described using one or more of these annotations. We find that the first two folds, the P-loop containing nucleotide triphosphate hydrolase and the NAD(P)-binding Rossmann-like homologous superfamilies, were α/β architectures responsible for introducing 35% (18/51) of the known mechanistic step types. We find that these two oldest structures in the phylogenomic analysis of protein domains introduced many mechanistic step types that were later combinatorially spread in catalytic history. The most common mechanistic step types included fundamental building blocks of enzyme chemistry: “Proton transfer,” “Bimolecular nucleophilic addition,” “Bimolecular nucleophilic substitution,” and “Unimolecular elimination by the conjugate base.” They were associated with the most ancestral fold structure typical of P-loop containing nucleotide triphosphate hydrolases. Over half of the mechanistic step types were introduced in the evolutionary timeline before the appearance of structures specific to diversified organisms, during a period of architectural diversification. The other half unfolded gradually after organismal diversification and during a period that spanned ∼2 billion years of evolutionary history.     

Molecular insights into the primary nucleation of polymorphic amyloid β dimers in DOPC lipid bilayer membrane

Alzheimer''s disease (AD) pathology is characterized by loss of memory cognitive and behavioral deterioration. One of the hallmarks of AD is amyloid β (Aβ) plaques in the brain that consists of Aβ oligomers and fibrils. It is accepted that oligomers, particularly dimers, are toxic species that are produced extracellularly and intracellularly in membranes. It is believed that the disruption of membranes by polymorphic Aβ oligomers is the key for the pathology of AD. This is a first study that investigate the effect of polymorphic “α‐helix/random coil” and “fibril‐like” Aβ dimers on 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) membrane. It has been found that the DOPC membrane promotes Aβ_1–42 “fibril‐like” dimers and impedes Aβ_1–42 “α‐helix/random coil” dimers. The N‐termini domains within Aβ_1–42 dimers play a role in Aβ aggregation in membrane milieus. In addition, the aromatic π–π interactions (involving residues F19 and F20 in Aβ_1–42) are the driving forces for the hydrophobic interactions that initiate the primary nucleation of polymorphic Aβ_1–42 dimers within DOPC membrane. Finally, the DOPC bilayer membrane thickness is locally decreased, and it is disrupted by an embedded distinct Aβ_1–42 dimer, due to relatively large contacts between Aβ_1–42 monomers and the DOPC membrane. This study reveals insights into the molecular mechanisms by which polymorphic early‐stage Aβ_1–42 dimers have distinct impacts on DOPC membrane.     

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed.     

The C-Terminal V5 Domain of Protein Kinase Cα Is Intrinsically Disordered,with Propensity to Associate with a Membrane Mimetic

The C-terminal V5 domain is one of the most variable domains in Protein Kinase C isoforms (PKCs). V5 confers isoform specificity on its parent enzyme through interactions with isoform-specific adaptor proteins and possibly through specific intra-molecular interactions with other PKC domains. The structural information about V5 domains in solution is sparse. The objective of this work was to determine the conformational preferences of the V5 domain from the α isoform of PKC (V5α) and evaluate its ability to associate with membrane mimetics. We show that V5α and its phosphorylation-mimicking variant, dmV5α, are intrinsically disordered protein domains. Phosphorylation-mimicking mutations do not alter the overall conformation of the polypeptide backbone, as evidenced by the local nature of chemical shift perturbations and the secondary structure propensity scores. However, the population of the “cis-trans” conformer of the Thr⁶³⁸-Pro⁶³⁹-Pro⁶⁴⁰ turn motif, which has been implicated in the down-regulation of PKCα via peptidyl-prolyl isomerase Pin1, increases in dmV5α, along with the conformational flexibility of the region between the turn and hydrophobic motifs. Both wild type and dmV5α associate with micelles made of a zwitterionic detergent, n-dodecylphosphocholine. Upon micelle binding, V5α acquires a higher propensity to form helical structures at the conserved “NFD” motif and the entire C-terminal third of the domain. The ability of V5α to partition into the hydrophobic micellar environment suggests that it may serve as a membrane anchor during the PKC maturation process.     

Ribosylation of the CD8αβ heterodimer permits binding of the nonclassical major histocompatibility molecule,H2-Q10

The CD8αβ heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD⁺, the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or β chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD⁺. These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses.     

Murine Cytomegalovirus m02 Gene Family Protects against Natural Killer Cell-Mediated Immune Surveillance

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient gamma(c)/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance.     

The Atomic Resolution Structure of Human AlkB Homolog 7 (ALKBH7), a Key Protein for Programmed Necrosis and Fat Metabolism

ALKBH7 is the mitochondrial AlkB family member that is required for alkylation- and oxidation-induced programmed necrosis. In contrast to the protective role of other AlkB family members after suffering alkylation-induced DNA damage, ALKBH7 triggers the collapse of mitochondrial membrane potential and promotes cell death. Moreover, genetic ablation of mouse Alkbh7 dramatically increases body weight and fat mass. Here, we present crystal structures of human ALKBH7 in complex with Mn(II) and α-ketoglutarate at 1.35 Å or N-oxalylglycine at 2.0 Å resolution. ALKBH7 possesses the conserved double-stranded β-helix fold that coordinates a catalytically active iron by a conserved HX(D/E) … X_n … H motif. Self-hydroxylation of Leu-110 was observed, indicating that ALKBH7 has the potential to catalyze hydroxylation of its substrate. Unlike other AlkB family members whose substrates are DNA or RNA, ALKBH7 is devoid of the “nucleotide recognition lid” which is essential for binding nucleobases, and thus exhibits a solvent-exposed active site; two loops between β-strands β6 and β7 and between β9 and β10 create a special outer wall of the minor β-sheet of the double-stranded β-helix and form a negatively charged groove. These distinct features suggest that ALKBH7 may act on protein substrate rather than nucleic acids. Taken together, our findings provide a structural basis for understanding the distinct function of ALKBH7 in the AlkB family and offer a foundation for drug design in treating cell death-related diseases and metabolic diseases.     

Molecular Interactions of Alzheimer's Biomarker FDDNP with Aβ Peptide

All-atom explicit solvent model and replica exchange molecular dynamics were used to investigate binding of Alzheimer''s biomarker FDDNP to the Aβ_10–40 monomer. At low and high concentrations, FDDNP binds with high affinity to two sites in the Aβ_10–40 monomer located near the central hydrophobic cluster and in the C-terminal. Analysis of ligand- Aβ_10–40 interactions at both concentrations identifies hydrophobic effect as a main binding factor. However, with the increase in ligand concentration the interactions between FDDNP molecules also become important due to strong FDDNP self-aggregation propensity and few specific binding locations. As a result, FDDNP ligands partially penetrate the core of the Aβ_10–40 monomer, forming large self-aggregated clusters. Ligand self-aggregation does not affect hydrophobic interactions as a main binding factor or the location of binding sites in Aβ_10–40. Using the Aβ_10–40 conformational ensemble in ligand-free water as reference, we show that FDDNP induces minor changes in the Aβ_10–40 secondary structure at two ligand concentrations studied. At the same time, FDDNP significantly alters the peptide tertiary fold in a concentration-dependent manner by redistributing long-range, side-chain interactions. We argue that because FDDNP does not change Aβ_10–40 secondary structure, its antiaggregation effect is likely to be weak. Our study raises the possibility that FDDNP may serve as a biomarker of not only Aβ fibril species, but of monomers as well.     

Model of β-Sheet of Muscle Fatty Acid Binding Protein of Locusta migratoria Displays Characteristic Topology

The β-sheet of muscle fatty acid binding protein of Locusta migratoria (Lm-FABP) was modeled by employing 2-D NMR data and the Rigid Body Assembly method. The model shows the β-sheet to comprise ten β-strands arranged anti-parallel to each other. There is a β-bulge between Ser 13 and Gln 14 which is a difference from the published structure of β-sheet of bovine heart Fatty Acid Binding Protein. Also, a hydrophobic patch consisting of Ile 45, Phe 51, Phe 64 and Phe 66 is present on the surface which is characteristic of most Fatty Acid Binding Proteins. A “gap” is present between βD and βE that provides evidence for the presence of a portal or opening between the polypeptide chains which allows ligand fatty acids to enter the protein cavity and bind to the protein.     

Highly Divergent T-cell Receptor Binding Modes Underlie Specific Recognition of a Bulged Viral Peptide bound to a Human Leukocyte Antigen Class I Molecule

Human leukocyte antigen (HLA)-I molecules can present long peptides, yet the mechanisms by which T-cell receptors (TCRs) recognize featured pHLA-I landscapes are unclear. We compared the binding modes of three distinct human TCRs, CA5, SB27, and SB47, complexed with a “super-bulged” viral peptide (LPEPLPQGQLTAY) restricted by HLA-B*35:08. The CA5 and SB27 TCRs engaged HLA-B*35:08^LPEP similarly, straddling the central region of the peptide but making limited contacts with HLA-B*35:08. Remarkably, the CA5 TCR did not contact the α1-helix of HLA-B*35:08. Differences in the CDR3β loop between the CA5 and SB27 TCRs caused altered fine specificities. Surprisingly, the SB47 TCR engaged HLA-B*35:08^LPEP using a completely distinct binding mechanism, namely “bypassing” the bulged peptide and making extensive contacts with the extreme N-terminal end of HLA-B*35:08. This docking footprint included HLA-I residues not observed previously as TCR contact sites. The three TCRs exhibited differing patterns of alloreactivity toward closely related or distinct HLA-I allotypes. Thus, the human T-cell repertoire comprises a range of TCRs that can interact with “bulged” pHLA-I epitopes using unpredictable strategies, including the adoption of atypical footprints on the MHC-I.