Marsupials are one of three major mammalian lineages that include the placental eutherians and the egg-laying monotremes. The marsupial brushtail possum is an important protected species in the Australian forest ecosystem. Molecules encoded by the MHC genes are essential mediators of adaptive immune responses in virus–host interactions. Yet, nothing is known about the peptide presentation features of any marsupial MHC class I (MHC I). This study identified a series of possum MHC I Trvu-UB*01:01 binding peptides derived from wobbly possum disease virus (WPDV), a lethal virus of both captive and feral possum populations, and unveiled the structure of marsupial peptide/MHC I complex. Notably, we found the two brushtail possum–specific insertions, the 3-aa Ile52Glu53Arg54 and 1-aa Arg154 insertions are located in the Trvu-UB*01:01 peptide binding groove (PBG). The 3-aa insertion plays a pivotal role in maintaining the stability of the N terminus of Trvu-UB*01:01 PBG. This aspect of marsupial PBG is unexpectedly similar to the bat MHC I Ptal-N*01:01 and is shared with lower vertebrates from elasmobranch to monotreme, indicating an evolution hotspot that may have emerged from the pathogen–host interactions. Residue Arg154 insertion, located in the α2 helix, is available for TCR recognition, and it has a particular influence on promoting the anchoring of peptide WPDV-12. These findings add significantly to our understanding of adaptive immunity in marsupials and its evolution in vertebrates. Our findings have the potential to impact the conservation of the protected species brushtail possum and other marsupial species.

Mammals are classified into three lineages: eutherians, marsupials, and monotremes (1). As nonplacental mammals, marsupials separated from eutherians ∼160 million years ago (2). Female marsupials have an external pouch, and immature young are raised there after birth until infancy. A wide variety of marsupials, ∼200 species, are found in Australia, New Guinea, and adjacent islands. In Australia, marsupials make up most of the native mammals. In addition to some large species, such as the wallaby, wombat, and koala, numerous smaller species, such as the brushtail possum, Tasmanian devil, sugar glider, and other typical Australian mammals are found (3). The separation of Australia from the continental plates resulted in geographical and, in turn, the animals’ reproductive isolation from those on the mainland. The evolution of reproductive isolation is the basis for the origin and maintenance of biological diversity (46), enabling marsupials to have exceptional reproductive development and immune characteristics.

The young marsupials are delivered from the uterus without immune organs or tissues, and their immune system develops outside the sterile range of the uterus; this characteristic makes them ideal models for developmental immunology research (7, 8). With the continuous advances in genomics (9), Igs, MHC genes, TCRs, and other marsupial immune-related genes have been identified; these genes are functionally similar to eutherians.

As a widely distributed marsupial native to Australia, the habitat of the brushtail possum (Trichosurus vulpecula) ranges from the temperate forests of Tasmania to the arid centers and the open tropical forests. However, since the middle of the nineteenth century, with the introduction to New Zealand of fur industry development, the population density of the brushtail possum has become much higher than its original distribution range and is now listed as a national pest in New Zealand. It is also a major wildlife host and vector of Mycobacterium bovis, causing damage to New Zealand dairy, beef, and venison industries (1012). Although wildlife diseases can be used as a biological control method to deal with invasive animals that threaten the local ecological environment and native species, they may threaten the survival of native species in a particular area and eventually become zoonosis channels (13). The brushtail possum is protected as an important species in the local forest ecosystem of Australia, but wobbly possum disease (WPD) is common in both captivity and free-living possum populations (14, 15). WPD is closely associated with WPD virus (WPDV) (16), which is classified as the family of Arteriviridae (17, 18). In the serological investigation of archived cases in New Zealand, it was found that the prevalence of WPD among free-living brushtail possum was ∼30% (19). An infected brushtail possum tends to show symptoms of a multifocal nervous system, often involving the vestibular system, resulting in ataxia, decreased climbing ability, and behavioral changes (16). There is currently no effective treatment, and most diseased possums will progress to death postinfection with WPDV, which affects the survival of this species to a certain extent. Studies of innate and adaptive immune responses are limited, except for a few studies on the humoral responses. The brushtail possum produced a detectable Ab response to rN protein after being infected with WPDV in experimental conditions. However, the protective effect of the Ab is still unknown (20). It was found that TNF-α can enhance the Ab response to Ags to a certain extent (21). The brushtail possum has the typical mammalian IL-2 gene, although its amino acid sequence homology is much lower than that of mice and humans (22). Nevertheless, the adaptive immune system in the brushtail possum is still less understood.

MHC class I (MHC I) molecules mediate the interaction between the virus and the host immune system by presenting peptides to T cells that lead to the initiation of an adaptive immune response (23). Previously, we determined the structure of the first bat MHC I complex from the Australia flying fox (Pteropus alecto). It was found that bat MHC I has a 3-aa or 5-aa insertion within their peptide binding groove (PBG), which had a specific influence on bat MHC I presentation peptide (24, 25). Interestingly, the characteristic of a 3-aa insertion is shared with most marsupials. It is unknown whether the insertion in marsupials suggests some immune characteristics similar to those of bats. Further studies need to be propelled to investigate the structure of marsupial MHC I, parallel to one of the bats.

Under selective pressures from pathogens and continually changing environments, MHC genes evolve rapidly in a pattern of “birth and death’’ to protect the host from the ever-changing pathogens (26, 27). Twenty-one MHC I genes are currently known for the brushtail possum. The first identified Trvu-UB*01:01 is presumed to be a classical class I gene (28). Among the 20 new sequences subsequently identified, Trvu01 to Trvu11 appear to be alleles of the previously described Trvu-UB*01:01 locus, and seven putative nonfunctional sequences (Trvu12-Trvu18) may derive from a pseudogene locus (29). Trvu19 and Trvu20 vary widely from the possum classical Trvu-UB*01:01. However, the molecular basis for the peptide binding and presentation by brushtail possum MHC I remains largely unknown.

In this study, we screened Trvu-UB*01:01-restricted peptides from GP3 of WPDV and visualized the structure of brushtail possum MHC I complexed with this peptide. To some extent, this study helps us to understand the special adaptive immunity of marsupials and provides insights to the structural similarities and differences between the brushtail possum and bat under the convergent evolution by the pressure of pathogens.

The cDNA amplification product coding the extracellular region of brushtail possum MHC I Trvu-UB*01:01 (AAK91168.1; GenBank) was cloned into pET-21a (+) vector (GENEWIZ, Suzhou, China). Mutation constructs of Trvu-UB*01:01 with Arg154 deletion (−1aa), with deletion of Ile52Glu53Arg54 (−3aa), E45M, and D59G were generated based on wild-type (WT) (Trvu-UB*01:01) and cloned into pET-21a (+) vector by PCR. To elucidate the function of Ile52Glu53Arg54 and Arg154 in Trvu-UB*01:01, the mutant Trvu-UB*01:01 with a deletion of 3-aa (Ile52Glu53Arg54) and 1-aa (Arg154) were synthesized, respectively. The mutant Trvu-UB*01:01 (D59G) was synthesized (Asp59 mutated into Gly59) to verify the interaction between Asp59 and Arg65 on the stability of Trvu-UB*01:01. To explore the influence of Glu45 on the His preference of B pocket, we prepared a mutant H chain of Trvu-UB*01:01 (E45M) (Glu45 mutated into Met45). The expression plasmid of human β2-microglobulin (β2m; residues 1–99) was constructed previously in our laboratory (30).

In this study, we used the WPDV-derived proteome to screen for potential binding peptides of Trvu-UB*01:01. Peptides WPDV-1 to WPDV-25 are derived from M, GP3, and NP in the WPDV proteome (M: GeneBank number AEU12354.1; GP3: GeneBank number AEU12350.1; NP: GeneBank number YP_009130640.1). Peptides HF-1 to HF-6 are derived from RdRp1ab and GP2 in the WPDV proteome (RdRp1ab: GeneBank number YP_009130631.2; GP2: GeneBank number YP_009130633.1). We used the NetMHCpan 4.0 server (Department of Health Technology, Lyngby, Denmark) (31) to predict peptides according to the binding score. Random peptide libraries (excluding cysteine) with lengths of 8, 9, and 10 aa were used in this study. The peptides were purified by high performance liquid chromatography and verified by mass spectrometry (MS) to a purity higher than 95%. The peptides were stored at −80°C in an ultralow-temperature refrigerator and dissolved in DMSO to a concentration of 100 μg/ml before use.

Trvu-UB*01:01, H chain mutants, and hβ2m were expressed in the form of inclusion bodies by the expression system of Escherichia coli. The washed and purified inclusion bodies were dissolved in 6 M guanidine hydrochloride at a final concentration of 30 mg/ml. hβ2m, peptide, and Trvu-UB*01:01 were then added into the refolding solution (0.1 M Tris-HCl [pH 8.0], 2 mM EDTA, 0.4 M l-arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione) (32) at the molar ratio of 1:3:1 in turn, then refolded at 4°C for 8 h (3335). After renaturation, the Trvu-UB*01:01/peptide complex was exchanged and concentrated into a solution containing 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl and then purified by a Superdex 200 Increase 10/300 GL column (GE Healthcare, Beijing, China).

Crystallization was carried out by sitting-drop vapor diffusion technique. Trvu-UB*01:01/peptide complex crystals were screened through Index Screen Kits, Crystal Screen/ Crystal Screen II Kits, and PEGRx 1 and PEGRx 2 Kits (Hampton Research). The plates were placed horizontally in 4°C and 18°C for about one month. The Trvu-UB*01:01/WPDV-12 crystals were grown in 0.1 M Na citrate trisbasic dihydrate (pH 5.0) and 30% v/v JEFFAMINE ED-2001 (pH 7.0). The single crystal was transferred to cryoprotective solutions containing 20% glycerol and then flash cooled and stored in liquid nitrogen until data collection. The x-ray diffraction data were collected at beamline BL19U of the Shanghai Synchrotron Radiation Facility (Shanghai, China).

High-resolution structural data were processed with the HKL-2000 software package (36). The structure of the Trvu-UB*01:01/WPDV-12 complex was resolved by molecular replacement using the Phaser MR program in CCP4. HLA-B*1501 structure (Protein Data Bank [PDB] code 5VZ5) was used as the replacement model (37). Coot (38) was used to build atomic models, and refinement was done with the refine program in Phenix (39). PyMOL and Coot were used to draw the MHC I structure and figure display.

The purified protein sample (Trvu-UB*01:01/peptide complex) was added with 2M acetic acid at a final concentration of 0.2 M. The mixture was placed in a water bath at 90°C for 5 min. The product was concentrated in a 3-kDa filter tube, and the peptides in the outer tube were collected. The eluted peptide concentration was determined, flash cooled, then stored at −80°C until use. The desalination of MHC peptides was performed on a C18 Solid Phase Extraction Column (Waters, Wilmslow, U.K.). The desalted peptides were separated on a Nano LC U3000 liquid phase system using an Acclaim Pepmap Nano C18 Trap column for peptide trapping and Acclaim Pepmap 100 C18 column for peptide separation. The hybrid Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used for MS data collection under the data-dependent acquisition mode, which enables individual peptide ions in MS1 scan to be isolated to generate fragment ions in MS2 scans for sequence determination. The resolution of MS1 and MS2 scans was set to 120 and 15K, respectively. The scanning range is ∼350–1800 m/z for the MS1 scan; the normalized collision energy is 27%. The dynamic exclusion time is 30 s.

MS data acquired in.raw format were transformed into .mgf format and were analyzed by peptide de novo sequencing algorithm pNovo+ (v3.1.3) (40) using the following parameter: fragmentation set to higher-energy collisional dissociation mode, no enzymatic cleavage, fixed modification was specified, oxidation in Met was set as variable modification, MS1 and MS2 mass errors were set to 7 ppm and 15 ppm, respectively. For each MS2 spectra, only the highest scoring sequence was kept. The sequences with length of 8, 9, and 10 aa containing no cysteine and an overall score >50 were included for downstream analysis. The sequence logo is presented using an online tool.

TA Instruments Nano DSC was used for testing the thermostabilities of brushtail possum MHC I Trvu-UB*01:01 and its corresponding mutants with a series of peptides. The concentration of the purified MHC I/peptide complexes was fixed into 1 mg/ml in 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl buffer solution. The volume of the TA Nano DSC calorimetry cell is 300 μl, and the minimum volume of the required sample was 600 μl. The buffer solution was first loaded into both the reference cell and the sample cell, and the background scanning was carried out at the rising rate of 1°C/min from 20°C to 90°C. The cleaning process is essential for the generation of credible and accurate differential scanning calorimetry (DSC) data. We followed the cleaning procedure recommended by the manufacturer. After cleaning, the protein sample was added to the sample well, and the protein buffer solution was loaded to the reference well. The endothermic or exothermic enthalpy from 20°C to 90°C was determined at the same rate of 1°C/min. A single endothermic peak is usually observed in the diagram. After calibration of instrumental baseline, transition baseline, and normalization to concentration, the peaks can be integrated to directly give measurements of enthalpy calorimetric pair process (ΔHcal) and melting temperature (Tm) (41). ΔH = T0T1Cp.dT. Data analysis was carried out by TA Instruments NanoAnalyze, which was matched with the instrument. The fitting data were exported and plotted using OriginPro9.1 software.

Through genetic analysis of the existing MHC I sequences of marsupials in GenBank, we found that these marsupial MHC I genes all have the same general typical MHC I domain as MHC I genes of other mammals. However, analysis of sequence revealed several unusual features of marsupials compared with eutherians and bats. In the previous structural analysis of bat Ptal-N*01:01, it is found that 3-aa or 5-aa insertion exists in the α1 helix in PBG. We also found the 3-aa insertion characterized in the MHC I sequence of marsupials (Fig. 1A, Supplemental Fig. 1). We took HLA-A*0201 as a reference sequence and followed the principle of not changing the sequence continuity. By comparing HLA-A*0201, Trvu-UB*01:01 also has a 1-aa Arg154 insertion between Ala150 and His151 (Fig. 1B). Without discriminating the number of amino acids that are inserted, we found that the insertion in α1 helix is present in 92.3% (12/13) brushtail possum MHC I genes, 100% (13/13) in the tammar wallaby, 75% (3/4) in the Tasmanian devil, 78.6% (11/14) in the opossum, 45% (9/20) in the koala, and 87.5% (49/56) in bats (Fig. 1C). However, the insertion is absent among all the MHC I molecules of eutherians, such as humans, mice, and rhesus.

FIGURE 1.

Amino acid insertion characteristics of marsupial MHC I. (A) Comparison of insertion in Trp51 and Val52 (compared with HLA-A*0201) of MHC I genes in marsupials (brushtail possum, tammar wallaby, Tasmanian devil, opossum, koala) and higher mammals (human, nonhuman primates, mice, horses, cattle, pigs, dogs, cats) and bats. Marsupials, bats, and higher mammals are distinguished by the orange, blue, and yellow rectangle. The red arrow represents the insertion sites. (B) Sequence alignment of amino acid insertions at the α2 helix of MHC I from marsupials and other representative mammals. (C and D) The proportion of MHC I genes in marsupials (brushtail possum, tammar wallaby, Tasmanian devil, opossum, koala), higher mammals, and bats inserted at Trp51 and Val52 (C) and Ala150 and His151 (D), respectively. Marsupials, bats, and higher mammals are distinguished by orange, blue, and yellow fan shapes, respectively. (E) The high proportion of positively charged amino acids as inserted residues in marsupials within the α2 helix.

FIGURE 1.

Amino acid insertion characteristics of marsupial MHC I. (A) Comparison of insertion in Trp51 and Val52 (compared with HLA-A*0201) of MHC I genes in marsupials (brushtail possum, tammar wallaby, Tasmanian devil, opossum, koala) and higher mammals (human, nonhuman primates, mice, horses, cattle, pigs, dogs, cats) and bats. Marsupials, bats, and higher mammals are distinguished by the orange, blue, and yellow rectangle. The red arrow represents the insertion sites. (B) Sequence alignment of amino acid insertions at the α2 helix of MHC I from marsupials and other representative mammals. (C and D) The proportion of MHC I genes in marsupials (brushtail possum, tammar wallaby, Tasmanian devil, opossum, koala), higher mammals, and bats inserted at Trp51 and Val52 (C) and Ala150 and His151 (D), respectively. Marsupials, bats, and higher mammals are distinguished by orange, blue, and yellow fan shapes, respectively. (E) The high proportion of positively charged amino acids as inserted residues in marsupials within the α2 helix.

Close modal

The previously identified canine MHC I also contains a 1-aa insertion within the α2 helix, but the insertion is Leu155 (42), different from marsupial MHC I. In marsupials, 100% of the koala (2/2) (only two complete sequences are available at this site), the opossum (14/14), and the Tasmanian devil (4/4) and 38.5% (5/13) of tammar wallaby and 84.6% (11/13) of brushtail possum MHC I have a 1-aa insertion (Fig. 1D). For the higher mammals in eutherians, such as humans, nonhuman primates, mice, and horses, no insertions are found in either the α1 or α2 helix. In terms of amino acid types inserted at position 154 in marsupials, Arg/Lys account for the majority (Fig. 1E), indicating a preference for positively charged amino acids within position 154 of marsupial MHC I.

In view of the significant effects of WPDV on the health and ecology of the marsupial brushtail possum, we screened the potential Trvu-UB*01:01 binding peptides from WPDV through online prediction and refolding (Table I). Eight of the twenty-five synthesized peptides can bind to Trvu-UB*01:01. Except for the WPDV-12, the remaining seven showed only weak binding capacity (Supplemental Fig. 2). The resolution of Trvu-UB*01:01–WPDV-12 hβ2m complex was 2.7 Å (Table II).

Table I.

Potential marsupial MHC I Trvu-UB*01:01 binding peptides

NameDerived ProteinPositionSequenceScoreaRefoldingb
WPDV-1 24–33 AFDTIIVAQP 0.02 − 
WPDV-2 26–35 DTIIVAQPML 0.0468 − 
WPDV-3 41–49 LITWKMFRL 0.15 − 
WPDV-4 45–54 MFRLIVTPV 0.1772 − 
WPDV-5 87–94 AIFTLTGV 0.0624 
WPDV-6 128–136 TSKVASEWP 0.0257 − 
WPDV-7 130–138 KVASEWPIL 0.1206 − 
WPDV-8 146–154 RTANGTHWP 0.0308 − 
WPDV-9 GP3 23–31 VLADNNTCL 0.1385 
WPDV-10 GP3 33–41 ATTISVDTL 0.0861 
WPDV-11 GP3 39–46 DTLCNVTL 0.0338 − 
WPDV-12 GP3 42–49 CNVTLNYP 0.026 ++ 
WPDV-13 GP3 75–82 LNYTSGNP 0.033 
WPDV-14 GP3 86–94 DAYHTARTL 0.151 
WPDV-15 GP3 99–106 DTVHVSFS 0.0138 − 
WPDV-16 GP3 104–112 SFSGHSFII 0.1561 − 
WPDV-17 GP3 119–127 FLSNTSVCI 0.1754 − 
WPDV-18 GP3 140–148 ITLYKSTGI 0.091 − 
WPDV-19 GP3 159–167 DFFISIANS 0.0285 − 
WPDV-20 GP3 177–185 LVICGFYMP 0.0369 − 
WPDV-21 GP3 181–189 GFYMPMVSL 0.104 
WPDV-22 NP 34–41 NYITPEQR 0.0205 − 
WPDV-23 NP 106–114 IIANDEAFV 0. 086 − 
WPDV-24 NP 108–117 ANDEAFVFAL 0.0673 
WPDV-25 NP 112–119 AFVFALTL 0.0789 − 
NameDerived ProteinPositionSequenceScoreaRefoldingb
WPDV-1 24–33 AFDTIIVAQP 0.02 − 
WPDV-2 26–35 DTIIVAQPML 0.0468 − 
WPDV-3 41–49 LITWKMFRL 0.15 − 
WPDV-4 45–54 MFRLIVTPV 0.1772 − 
WPDV-5 87–94 AIFTLTGV 0.0624 
WPDV-6 128–136 TSKVASEWP 0.0257 − 
WPDV-7 130–138 KVASEWPIL 0.1206 − 
WPDV-8 146–154 RTANGTHWP 0.0308 − 
WPDV-9 GP3 23–31 VLADNNTCL 0.1385 
WPDV-10 GP3 33–41 ATTISVDTL 0.0861 
WPDV-11 GP3 39–46 DTLCNVTL 0.0338 − 
WPDV-12 GP3 42–49 CNVTLNYP 0.026 ++ 
WPDV-13 GP3 75–82 LNYTSGNP 0.033 
WPDV-14 GP3 86–94 DAYHTARTL 0.151 
WPDV-15 GP3 99–106 DTVHVSFS 0.0138 − 
WPDV-16 GP3 104–112 SFSGHSFII 0.1561 − 
WPDV-17 GP3 119–127 FLSNTSVCI 0.1754 − 
WPDV-18 GP3 140–148 ITLYKSTGI 0.091 − 
WPDV-19 GP3 159–167 DFFISIANS 0.0285 − 
WPDV-20 GP3 177–185 LVICGFYMP 0.0369 − 
WPDV-21 GP3 181–189 GFYMPMVSL 0.104 
WPDV-22 NP 34–41 NYITPEQR 0.0205 − 
WPDV-23 NP 106–114 IIANDEAFV 0. 086 − 
WPDV-24 NP 108–117 ANDEAFVFAL 0.0673 
WPDV-25 NP 112–119 AFVFALTL 0.0789 − 
a

The score values were automatically displayed while predicting potential binding peptides; http://www.cbs.dtu.dk/services/NetMHCpan/.

b

Peptides that are capable of assisting Trvu-UB*01:01 H chain to renature with human β2m are indicated with “+.” Peptides that cannot assist Trvu-UB*01:01 to renature with human β2m are indicated with “−.” “++” represents a strong binding, and “+” indicates weak binding.

Table II.

X-ray diffraction data collection and refinement statistics

ParameterTrvu-UB*01:01/WPDV-12
PDB code 7EDO 
Peptide sequence CNVTLNYP 
Data processing  
 Space group I23 
 Cell parameter (Å)  
 a, b, c (Å) 193.63, 193.63, 193.63 
 α, β, γ (o90.00, 90.00, 90.00 
 Wavelength (Å) 0.97918 
 Resolution (Å) 50-2.7 (2.8–2.7) 
 Total reflections 1315530 
 Unique reflections 33061 
 Completeness (%)a 99.88 (99.91) 
 Redundancy 38.8 (39.8) 
 Rmerge (%)b 12.9 (87.7) 
I/σ 2.69 
Refinement  
 Rwork (%) 18.16 
 Rfree (%) 23.74 
 RMSDc  
  Bonds (Å) 0.009 
  Angle (o1.31 
  Average B factor (Å240.88 
Ramachandran plot quality (%)  
 Favored (%) 94.33 
 Allowed (%) 5.14 
 Outliers (%) 0.53 
ParameterTrvu-UB*01:01/WPDV-12
PDB code 7EDO 
Peptide sequence CNVTLNYP 
Data processing  
 Space group I23 
 Cell parameter (Å)  
 a, b, c (Å) 193.63, 193.63, 193.63 
 α, β, γ (o90.00, 90.00, 90.00 
 Wavelength (Å) 0.97918 
 Resolution (Å) 50-2.7 (2.8–2.7) 
 Total reflections 1315530 
 Unique reflections 33061 
 Completeness (%)a 99.88 (99.91) 
 Redundancy 38.8 (39.8) 
 Rmerge (%)b 12.9 (87.7) 
I/σ 2.69 
Refinement  
 Rwork (%) 18.16 
 Rfree (%) 23.74 
 RMSDc  
  Bonds (Å) 0.009 
  Angle (o1.31 
  Average B factor (Å240.88 
Ramachandran plot quality (%)  
 Favored (%) 94.33 
 Allowed (%) 5.14 
 Outliers (%) 0.53 
a

Data completeness represents the ratio of the number of independent reflections to the total theoretical number.

b

Rmerge = Σhkl Σi|Ii−〈I〉| Σhkl ΣiIi, where Ii is the integrated intensity of the reflections.

c

R = Σhkl ǁFobs|−k |Fcall ǁ/ Σhkl|Fobs|. Rfree is calculated for a subset (5%) of reflections, and Rwork is calculated for the remaining 95% of reflections used for refinement.

Trvu-UB*01:01 shows a consistent overall characteristic compared with known MHC I structures in other mammals. The H chain of the Trvu-UB*01:01 folds into three domains. The α1 and α2 domains constitute the classical PBG, which contains two α helices and eight-strand β folds, whereas the α3 domain and β2m exhibit typical Ig-like structures and provide support for the PBG (Fig. 2A). Two molecules exist in one asymmetric unit of the Trvu-UB*01:01/WPDV-12 structure, molecule 1 (M1) and molecule 2 (M2). M1 and M2 have a generally similar conformation with a root mean square difference (RMSD) of 1.0 Å (0.9 Å of the two H chains, 0.4 Å of the β2m, and 0.3 Å of two peptides) (Fig. 2A, Supplemental Fig. 3A–D). Trvu-UB*01:01 shows a lower RMSD with bat MHC I (0.7 Å) compared with human HLA-A*0201(1.1 Å) (Fig. 2B). Furthermore, similar to α1 helix of Ptal-N*01:01, the α1-helical extension of the N-terminal of PBG also exists in the structure of Trvu-UB*01:01. Meanwhile, the other apparent structural feature of Trvu-UB*01:01 distinguished to HLA-A*0201 and Ptal-N*01:01 is located in the α2 helix. Trvu-UB*01:01 has a longer extension of the α2 helix than MHC I from other mammals (Fig. 2C).

FIGURE 2.

The overall structure of peptides binding to possum MHC I Trvu-UB*01:01. (A) The overall structure of Trvu-UB*01:01. The contrast of the global conformation of two molecules in one asymmetric unit of Trvu-UB*01:01 structure. M1 is shown in blue-white and M2 is shown in deep blue. The WPDV-derived peptide WPDV-12 in PBG is shown in blue-white (M1) and deep blue (M2), respectively. (B) Trvu-UB*01:01 superimposed with MHC I structures in other mammals. Human HLA-A*0201 (PDB number 3I6G) is shown in light magenta, bat Ptal-N*01:01 (PDB number 6J2D) is shown in yellow, and Trvu-UB*01:01 is shown in blue-white. (C) The different conformations between the α1 and α2 helices of Trvu-UB*01:01 (blue-white) and human HLA-A:0201 (light magenta) and bat Ptal-N*01:01 (yellow) are shown in black rectangles.

FIGURE 2.

The overall structure of peptides binding to possum MHC I Trvu-UB*01:01. (A) The overall structure of Trvu-UB*01:01. The contrast of the global conformation of two molecules in one asymmetric unit of Trvu-UB*01:01 structure. M1 is shown in blue-white and M2 is shown in deep blue. The WPDV-derived peptide WPDV-12 in PBG is shown in blue-white (M1) and deep blue (M2), respectively. (B) Trvu-UB*01:01 superimposed with MHC I structures in other mammals. Human HLA-A*0201 (PDB number 3I6G) is shown in light magenta, bat Ptal-N*01:01 (PDB number 6J2D) is shown in yellow, and Trvu-UB*01:01 is shown in blue-white. (C) The different conformations between the α1 and α2 helices of Trvu-UB*01:01 (blue-white) and human HLA-A:0201 (light magenta) and bat Ptal-N*01:01 (yellow) are shown in black rectangles.

Close modal

The peptide WPDV-12 presented by Trvu-UB*01:01 has a clear electron density. The 8-mer peptide exhibits an M-shaped arched conformation in the PBG (Fig. 3A) with the second (P2), fifth (P5), and eighth (PΩ) residues protruding into the B, C, and F pockets, respectively. To understand the role of these anchor residues in the Trvu-UB*01:01 binding motif, the P2, P5, and PΩ amino acids of WPDV-12 were mutated into Gly, and the thermal stabilities of Trvu-UB*01:01 complexed to these peptide mutants, WPDV-12-N2G, WPDV-12-L5G, and WPDV-12-P8G, were detected by DSC. The Tm of peptide WPDV-12 with PΩ mutation is the lowest (Tm = 39.87°C), whereas the P2 mutant WPDV-12-N2G also possesses a weaker binding capacity with Trvu-UB*01:01 (Tm = 50.25°C). Notably, the protein stability decreases significantly after substitution at P5 (Tm = 40.84°C) compared with WT WPDV-12 (Tm = 59.39°C) (Fig. 3B). Generally, the P5 residue of the peptide plays a secondary anchor as a traditional definition in MHC I binding peptides (43). Given the vital role of the P5 residue in the stability of Trvu-UB*01:01/WPDV-12, it is necessary to verify whether the P5 residue acts as the primary anchor as PΩ in the peptide presented in Trvu-UB*01:01. Thus, we predicted and synthesized another Trvu-UB*01:01 binding peptide, HF-2 (THVTGTPTF), and its mutant peptides HF-2-H2G, HF-2-T6G, and HF-2-F9G, mutating at the P2, P6, and PΩ positions. The fifth position of HF-2 is glycine, but the peptide exhibits the strongest binding capacity with Trvu-UB*01:01 (Tm = 62.96°C) compared with HF-2-T6G, HF-2-H2G, and HF-2-F9G (Fig. 3C). The P6 mutant HF-2-T6G shows relatively stable binding ability, and the Tm is 57.30°C. We found that the peptide HF-2-H2G and peptide HF-2-P8G have the similar but weakest binding capacity, with Tms of 37.74°C and 37.73°C, respectively. Therefore, we can see that P2 and PΩ still act as the primary anchor residues of the peptides binding and P5 or P6 act as the secondary anchor residues. In the C pocket, residues Tyr9, Phe77, and Tyr102 form a hydrophobic space, which prefers the hydrophobic side chain of P5-Leu because of hydrophobicity (Fig. 3D). In other words, the importance of the P5 amino acid in WPDV-12 for binding is only shown in this peptide.

FIGURE 3.

The conformation of the peptide presented by Trvu-UB*01:01. (A) The electron density map of peptide WPDV-12 presented by Trvu-UB*01:01. (B) DSC was used to detect the thermal stabilities of Trvu-UB*01:01 complexed with peptide WPDV-12 and its mutations (i.e., WPDV-12-N2G, WPDV-12-L5G, and WPDV-12-P8G). (C) DSC was used to detect the thermal stabilities of peptide HF-2, HF-2-H2G, HF-2-T6G, and HF-2-F9G after mutation. (D) Amino acid composition of the Trvu-UB*01:01 C pocket (cyan sticks) anchored by P5-Leu of peptide WPDV-12.

FIGURE 3.

The conformation of the peptide presented by Trvu-UB*01:01. (A) The electron density map of peptide WPDV-12 presented by Trvu-UB*01:01. (B) DSC was used to detect the thermal stabilities of Trvu-UB*01:01 complexed with peptide WPDV-12 and its mutations (i.e., WPDV-12-N2G, WPDV-12-L5G, and WPDV-12-P8G). (C) DSC was used to detect the thermal stabilities of peptide HF-2, HF-2-H2G, HF-2-T6G, and HF-2-F9G after mutation. (D) Amino acid composition of the Trvu-UB*01:01 C pocket (cyan sticks) anchored by P5-Leu of peptide WPDV-12.

Close modal

When we compared the MHC I sequence of brushtail possum with other vertebrate mammals, we found that Trvu-UB*01:01 also has an Arg154 insertion at the α2 helix (between residues Ala150 and His151 of HLA-A*0201). Compared with HLA-A*0201, the Arg154 insertion resulted in an additional half-turn of the α2 helix in the structure, and the measured distance between the two structures at the most expanded place is 4.0 Å (Fig. 4A). It can be observed that Arg154 is located at the highest site of Trvu-UB*01:01 (Fig. 4B). We analyzed M1 and M2 and found that the inserted two arginines have an individual difference in the side chain’s conformation. The side chain of Arg154 in M1 is high upward, whereas Arg154 in M2 is downward (Fig. 4C).

FIGURE 4.

Unique Arg154 insertion in brushtail possum MHC I for the impact on peptide presentation. (A) Conformational changes resulting from the insertion of Trvu-UB*01:01 at the α2 helix. After being superimposed, all atoms of Trvu-UB*01:01 (orange) and HLA-A*0201 (light blue), with a predicted distance of 4.0 Å at the extended α2 helix (between a Cα atom of Ser155 of Trvu-UB*01:01 and His151 of HLA-A*0201) are shown. (B) Comparison of Trvu-UB*01:01 M1 (orange) and M2 (cyan) structures. The two molecules are aligned according to the α1 helix. The gray arrow is used to highlight the insertion site. (C) The different conformations of M1 and M2 in Arg154 were inserted in the α2 helix. The inserted Arg154 shows the side chain of residues in stick form, with M1 shown in orange and M2 shown in cyan. (D and E) Effect of Arg154 deletion (−1aa) on peptide presentation. The effect of Arg154 deficiency on peptide presentation was characterized by in vitro renaturation ability (D) and DSC (E), respectively. (F) Solvent-accessible surface area between WPDV-12 and Trvu-UB*01:01. The part that is completely exposed to the solvent is shown in blue, and the part where there is a potential interaction between the two is colored from blue to orange according to the strength of the force. (G) Structural characterization of Arg154 insertion for peptide presentation. Trvu-UB*01:01 is superimposed on the HLA-A*0201. (H) Simulated structure diagram of HLA-A*0201 (Val152 mutated into Ile). Val152 (light blue), Ile156 (orange), and P6-Asn (orange) are shown in side-chains as stick form. The distance between OD1 and N atoms of WPDV-12 P6-Asn and Ile are indicated in red and blue dotted lines, respectively.

FIGURE 4.

Unique Arg154 insertion in brushtail possum MHC I for the impact on peptide presentation. (A) Conformational changes resulting from the insertion of Trvu-UB*01:01 at the α2 helix. After being superimposed, all atoms of Trvu-UB*01:01 (orange) and HLA-A*0201 (light blue), with a predicted distance of 4.0 Å at the extended α2 helix (between a Cα atom of Ser155 of Trvu-UB*01:01 and His151 of HLA-A*0201) are shown. (B) Comparison of Trvu-UB*01:01 M1 (orange) and M2 (cyan) structures. The two molecules are aligned according to the α1 helix. The gray arrow is used to highlight the insertion site. (C) The different conformations of M1 and M2 in Arg154 were inserted in the α2 helix. The inserted Arg154 shows the side chain of residues in stick form, with M1 shown in orange and M2 shown in cyan. (D and E) Effect of Arg154 deletion (−1aa) on peptide presentation. The effect of Arg154 deficiency on peptide presentation was characterized by in vitro renaturation ability (D) and DSC (E), respectively. (F) Solvent-accessible surface area between WPDV-12 and Trvu-UB*01:01. The part that is completely exposed to the solvent is shown in blue, and the part where there is a potential interaction between the two is colored from blue to orange according to the strength of the force. (G) Structural characterization of Arg154 insertion for peptide presentation. Trvu-UB*01:01 is superimposed on the HLA-A*0201. (H) Simulated structure diagram of HLA-A*0201 (Val152 mutated into Ile). Val152 (light blue), Ile156 (orange), and P6-Asn (orange) are shown in side-chains as stick form. The distance between OD1 and N atoms of WPDV-12 P6-Asn and Ile are indicated in red and blue dotted lines, respectively.

Close modal

To further investigate the effect of the Arg154 insertion on peptide presentation, we constructed a mutation with Arg154 deletion (−1aa) at the α2 helix of Trvu-UB*01:01. The binding ability of Trvu-UB*01:01 (−1aa) to the peptide is significantly weaker than that of Trvu-UB*01:01 (WT), according to the results of refolding (Fig. 4D). In line with the renaturation, the DSC results showed that the thermostability of the Trvu-UB*01:01 (−1aa)/WPDV-12 (Tm = 54.85°C) complex is decreased compared with that of WT (Tm = 60.30°C) (Fig. 4E), indicating that Arg154 insertion does affect peptide presentation.

Then we analyzed the mechanism of this phenomenon from the structure, which shows a concordant molecular basis for the peptide presentation impacted by Arg154 insertion within α2 helix. Structurally, Ile156 surrounding Arg154 is found to be close to P6-Asn. In terms of interaction force and solvent-accessible surface area, not only does the van der Waals force exist between Ile156 and P6-Asn (Table III), but compared with the part completely exposed to the solvent (blue), P6-Asn and Ile156 interact with each other from the contact interface (Fig. 4F). Using HLA-A*0201 as a reference, we found that Ile156 (Trvu-UB*01:01) is structurally corresponding to Val152 (HLA-A*0201). If Val152 is defined as a horizontal toward the PBG, the residue of Trvu-UB*01:01 Ile156 is slightly upward (Fig. 4G). The side chain of residue Ile156 of Trvu-UB*01:01 creates a 3.6-Å distance from P6 of WPDV-12 (Fig. 4G). If we assume that the α2 helix of HLA-A*0201 is the mutated structure after deletion of Arg154 by Trvu-UB*01:01, then the Ile156 of Trvu-UB*01:01 will move to the Val152 site of HLA-A*0201. We simulated the structure of HLA-A*0201 (V152I) mutation to represent the absence of Arg154 in Trvu-UB*01:01 and calculated the OD1 and N atoms’ distance of Ile152 from P6-Asn to be 3.5 Å and 3.3 Å, respectively (Fig. 4H). Thus, steric hindrance caused by the closer distance might be formed between Ile and P6-Asn of WPDV-12, whereas the insertion of Arg154 may increase the space for peptide binding to some extent, thus promoting peptide anchoring.

Table III.

Hydrogen bonds and van der Waals interactions between the WPDV-12 and Trvu-UB*01:01 H chain

ComplexPeptideHydrogen BondVan der Waals Contact Residues
ResidueAtomResidueAtom
Trvu-UB*01:01/β2m/WPDV-12 P1-Cys Tyr7 Tyr175 OH
OH 
Tyr7, Tyr163, Tyr171 
 Tyr163 OH  

P2-Asn 

Gln66
Glu45 
OE1
OE1
OE2 
Tyr7, Tyr9, Gln66, Ile69, Leu70, Tyr102 
  OH  
 OD1 Tyr9 OH  
P3-Val Tyr102 OH Ile69, Asn73, Tyr163 
 Asn73 ND2  
P4-Thr   Ile69 
P5-Leu Asn73 OD1 Tyr9, Asn73, Trp160 
P6-Asn   Val76, Ile156 
 OD1    
P7-Tyr Trp150 NE1  
P8-Pro Thr146
Tyr87 
OG1
OH 
Gly80, Thr83, Thr146, Lys149 
  OXT Lys149 NZ 
ComplexPeptideHydrogen BondVan der Waals Contact Residues
ResidueAtomResidueAtom
Trvu-UB*01:01/β2m/WPDV-12 P1-Cys Tyr7 Tyr175 OH
OH 
Tyr7, Tyr163, Tyr171 
 Tyr163 OH  

P2-Asn 

Gln66
Glu45 
OE1
OE1
OE2 
Tyr7, Tyr9, Gln66, Ile69, Leu70, Tyr102 
  OH  
 OD1 Tyr9 OH  
P3-Val Tyr102 OH Ile69, Asn73, Tyr163 
 Asn73 ND2  
P4-Thr   Ile69 
P5-Leu Asn73 OD1 Tyr9, Asn73, Trp160 
P6-Asn   Val76, Ile156 
 OD1    
P7-Tyr Trp150 NE1  
P8-Pro Thr146
Tyr87 
OG1
OH 
Gly80, Thr83, Thr146, Lys149 
  OXT Lys149 NZ 

As with bat Ptal-N*01:01, Trvu-UB*01:01 3-aa Ile52Glu53Arg54 are inserted at the N terminus of the α1 helix and show α1 helix extension of the PBG but different from human HLA-A*0201 (Fig. 5A). In Trvu-UB*01:01, the pairing relationship between Asp59 and Arg65, like bat Ptal-N*01:01, can also be found (Fig. 5B). In Ptal-N*01:01, the insertion of 3-aa renders Asp59 closer to the N-terminal of the binding peptide. Asp59, Arg65, and P1-Asp of the peptide form a stable hydrogen network (Fig. 5C). However, Asp59 and Arg65 in Trvu-UB*01:01 do not form any polar contacts with the P1-Cys of peptide WPDV-12 and only form salt bridges within Asp59 and Arg65. In addition, after being superimposed with Ptal-N*01:01, we found that the residues of Trvu-UB*01:01 Asp59 and Arg65 display position shift with longer distances from the P1-C (Fig. 5D). To determine whether the preference of peptide P1-D by bat Ptal-N*01:01 can be extended to Trvu-UB*01:01, we conducted P1-C into C1D of peptide WPDV-12 and found that, compared with WPDV-12 (Tm = 60.37°C), there was no increase in the thermal stability of peptide WPDV-12-C1D (Tm = 55.70°C) (Fig. 5E). In conclusion, it is difficult for Asp59, Arg65, and WPDV-12-P1C to form a stable hydrogen network like Ptal-N*01:01.

FIGURE 5.

Insertion of 3-aa Ile52Glu53Arg54 stabilized the N terminus of Trvu-UB*01:01, in parallel with bat Ptal-N*01:01. (A) Superimposing of N terminus peptide binding grooves (PBGs) from Trvu-UB*01:01 (cyan), Ptal-N*01:01 (green), HLA-A*0201 (light magenta). (B) Salt bridges between Trvu-UB*01:01/WPDV-12 complex D59 and R65 residues. (C) Hydrogen bond network of A pocket of Ptal-N*01:01. (D) Superimposition of Trvu-UB*01:01/WPDV-12 (cyan) and Ptal-N*01:01/HeV1 (green) structures. The conformational differences of D59 and R65 residues and peptide P1 site in Trvu-UB*01:01 and Ptal-N*01:01 structures are indicated by bright yellow, blue, and orange double arrows, respectively. (E) DSC assessed the ability of Trvu-UB*01:01 to present peptides WPDV-12 and WPDV-12-C1D. (F and G) The abilities of peptide WPDV-12 presented by Trvu-UB*01:01 and Trvu-UB*01:01 (−3aa) were evaluated using in vitro refolding (F) and DSC (G). (H) The abilities of both the Trvu-UB*01:01 (D59G) mutant and Trvu-UB*01:01 (WT) to present peptide WPDV-12 were assessed by DSC.

FIGURE 5.

Insertion of 3-aa Ile52Glu53Arg54 stabilized the N terminus of Trvu-UB*01:01, in parallel with bat Ptal-N*01:01. (A) Superimposing of N terminus peptide binding grooves (PBGs) from Trvu-UB*01:01 (cyan), Ptal-N*01:01 (green), HLA-A*0201 (light magenta). (B) Salt bridges between Trvu-UB*01:01/WPDV-12 complex D59 and R65 residues. (C) Hydrogen bond network of A pocket of Ptal-N*01:01. (D) Superimposition of Trvu-UB*01:01/WPDV-12 (cyan) and Ptal-N*01:01/HeV1 (green) structures. The conformational differences of D59 and R65 residues and peptide P1 site in Trvu-UB*01:01 and Ptal-N*01:01 structures are indicated by bright yellow, blue, and orange double arrows, respectively. (E) DSC assessed the ability of Trvu-UB*01:01 to present peptides WPDV-12 and WPDV-12-C1D. (F and G) The abilities of peptide WPDV-12 presented by Trvu-UB*01:01 and Trvu-UB*01:01 (−3aa) were evaluated using in vitro refolding (F) and DSC (G). (H) The abilities of both the Trvu-UB*01:01 (D59G) mutant and Trvu-UB*01:01 (WT) to present peptide WPDV-12 were assessed by DSC.

Close modal

We also constructed a mutant with −3aa to investigate the role of 3-aa Ile52Glu53Arg54 insertion in the peptide binding with Trvu-UB*01:01. It is elucidated that Trvu-UB*01:01 (−3aa) can still renature in the presence of WPDV-12, but the renaturation efficiency is significantly reduced compared with Trvu-UB*01:01 (WT) (Fig. 5F). At the same time, the thermal stability of the complex detected by DSC can help prove that the Tm of Trvu-UB*01:01 (−3aa) (Tm = 43.15°C) is much lower than that of Trvu-UB*01:01 (WT) (Tm = 60.24°C) (Fig. 5G), which indicates that the binding capacity of Trvu-UB*01:01 (−3aa) with peptide is decreased.

By the pushing of 3-aa insertion, Asp59 in the A pocket of Trvu-UB*01:01 forms the salt bridges with Arg65. To investigate the impact of the salt bridges on the peptide presentation and stability of Trvu-UB*01:01, we carried out mutation against Trvu-UB*01:01 at Asp59 (i.e., Trvu-UB*01:01 [D59G]) and found that the Tm of Trvu-UB*01:01 (D59G)/WPDV-12 complex (Tm = 55.08°C) is lower than that of Trvu-UB*01:01/WPDV-12 (Tm = 60.09°C) after destroying the interact forces formed by the Asp59 and Arg65 (Fig. 5H). Therefore, the 3-aa Ile52Glu53Arg54 insertion of Trvu-UB*01:01 and the pairing of Asp59 and Arg65 caused by the insertion may have a significant effect on maintaining the stability of the N terminus of Trvu-UB*01:01.

To rapidly and effectively identify the binding motif of Trvu-UB*01:01, we performed peptide sequencing using liquid chromatography–tandem MS (LC-MS/MS) by analyzing the eluted peptides from the Trvu-UB*01:01 renatured with random 8-mer, 9-mer, and 10-mer peptide pools. LC-MS/MS detected a total of 5953 peptides among the Trvu-UB*01:01-binding 9-mer peptides, and the sequence logo was mapped using the WebLogo online tool (44, 45). P2 in the sequence logo shows a strong preference for histidine together with Phe, Leu, and Val (Fig. 6A–C). To examine whether LC-MS/MS results are reliable, we selected six peptides (Table IV) from LC-MS/MS–analyzed 9-mer results, according to optimal anchor residues of the Trvu-UB*01:01 binding peptides from WPDV (Table I). The six WPDV-derived peptides all assist the folding of Trvu-UB*01:01 and β2m to form a natural conformation (Fig. 6D), illustrating that Trvu-UB*01:01 can use P2-H, PΩ-F as its optimal anchor motif, in addition to the P2-N and PΩ-P in the structural determined peptide WPDV-12.

FIGURE 6.

Histidine as a rare preference for P2 anchoring in Trvu-UB*01:01 binding peptides. (AC) Sequence logo of LC-MS/MS de novo analysis. The sequence logos show the preference of Trvu-UB*01:01 for 8-mer (A), 9-mer (B), and 10-mer (C) with specific motifs from P1 to PΩ. The logo is presented using WebLogo online tool (http://weblogo.berkeley.edu/). The sequences of eluted peptides can be found in Supplemental Table I. (D) The binding capabilities of six peptides presented by Trvu-UB*01:01 were evaluated by in vitro refolding. (E) The interacting forces in the B pocket of Trvu-UB*01:01/WPDV-12. Residues Glu45, Gln66, and Tyr9 are indicated in cyan, and P2-Asn of WPDV-12 are indicated in blue, respectively. The hydrogen bonds are indicated in purple dotted lines. (F and G) The vacuum electrostatic surface potential for B pocket in Trvu-UB*01:01 (F) and HLA-A*0201 (G). The side chains of residue Glu45 in Trvu-UB*01:01 and residue Met45 in HLA-A*0201 are shown as sticks. (H) DSC evaluated the thermal stabilities of peptide WPDV-12-N2H (the second amino acid of WPDV-12 change into His) with Trvu-UB*01:01 (WT) and Trvu-UB*01:01 (E45M) mutant.

FIGURE 6.

Histidine as a rare preference for P2 anchoring in Trvu-UB*01:01 binding peptides. (AC) Sequence logo of LC-MS/MS de novo analysis. The sequence logos show the preference of Trvu-UB*01:01 for 8-mer (A), 9-mer (B), and 10-mer (C) with specific motifs from P1 to PΩ. The logo is presented using WebLogo online tool (http://weblogo.berkeley.edu/). The sequences of eluted peptides can be found in Supplemental Table I. (D) The binding capabilities of six peptides presented by Trvu-UB*01:01 were evaluated by in vitro refolding. (E) The interacting forces in the B pocket of Trvu-UB*01:01/WPDV-12. Residues Glu45, Gln66, and Tyr9 are indicated in cyan, and P2-Asn of WPDV-12 are indicated in blue, respectively. The hydrogen bonds are indicated in purple dotted lines. (F and G) The vacuum electrostatic surface potential for B pocket in Trvu-UB*01:01 (F) and HLA-A*0201 (G). The side chains of residue Glu45 in Trvu-UB*01:01 and residue Met45 in HLA-A*0201 are shown as sticks. (H) DSC evaluated the thermal stabilities of peptide WPDV-12-N2H (the second amino acid of WPDV-12 change into His) with Trvu-UB*01:01 (WT) and Trvu-UB*01:01 (E45M) mutant.

Close modal
Table IV.

The eluted peptides from Trvu-UB*01:01 with P2-H and PΩ-F/L anchors

NameDerived ProteinPositionSequenceScoreaRefoldingb
HF-1 GP2 190–198 IHAFLRLRF 0.3196 ++ 
HF-2 RdRp1ab 1413–1421 THVTGTPTF 0.2879 ++ 
HF-3 RdRp1ab 2093–2101 GHEWDFEEL 0.0265 
HF-4 RdRp1ab 2298–2306 DHKVYMFNL 0.0407 ++ 
HF-5 RdRp1ab 2412–2420 YHRHCVSEF 0.3039 ++ 
HF-6 RdRp1ab 3180–3188 CHHVTSPNL 0.0507 ++ 
NameDerived ProteinPositionSequenceScoreaRefoldingb
HF-1 GP2 190–198 IHAFLRLRF 0.3196 ++ 
HF-2 RdRp1ab 1413–1421 THVTGTPTF 0.2879 ++ 
HF-3 RdRp1ab 2093–2101 GHEWDFEEL 0.0265 
HF-4 RdRp1ab 2298–2306 DHKVYMFNL 0.0407 ++ 
HF-5 RdRp1ab 2412–2420 YHRHCVSEF 0.3039 ++ 
HF-6 RdRp1ab 3180–3188 CHHVTSPNL 0.0507 ++ 
a

The score values were automatically displayed while predicting potential binding peptides; http://www.cbs.dtu.dk/services/NetMHCpan/.

b

Peptides that are capable of assisting Trvu-UB*01:01 H chain to renature with human β2m are indicated with “+.” “++” represents a strong binding, and “+” indicates weak binding.

In the structure of Trvu-UB*01:01/WPDV-12, the side chain of P2-Asn forms hydrogen bonds with Gln66, Glu45, and Tyr9, respectively, to stabilize the conformation (Fig. 6E). Further analysis of the residues that make up the B pocket shows that Glu45 lies at the bottom of the pocket, making the B pocket negatively charged (Fig. 6F). For other species, such as the human HLA-A*0201 B pocket, the amino acid at position 45 is Met, and the residue corresponding to this site in most mammals is Met or other noncharged amino acids such as Ala (Fig. 6G). Therefore, the charge attraction by Glu45 may cause the preference of B pocket for His. To further verify the function of Glu45 on the peptide motif of Trvu-UB*01:01 binding peptides, we mutated the Glu45 from a negatively charged residue to an uncharged Met, as in HLA-A*0201, and attempted to verify the effect through DSC (Fig. 6H). The Tm of the Trvu-UB*01:01 (E45M)/WPDV-12-N2H complex (Tm = 50.33°C) is significantly reduced compared with that of the WT Trvu-UB*01:01/WPDV-12-N2H complex (Tm = 65.35°C), which indicates the mutation had a lower binding capacity.

The pathogens among wildlife host groups temporarily pose no direct threat to human health or livestock and are often neglected but pivotal. In Australia, most representative marsupials are frequently threatened by bacterial (46), viral (4750), and neoplastic diseases (51, 52). Like bats, marsupials may have evolved some characteristics independently because of geographic and reproductive isolation. MHC Is are essential mediators in the interaction between the virus and the host immune system (53). To our knowledge, our study reveals the first structure of the marsupial MHC I Trvu-UB*01:01 from brushtail possum complexed with WPDV-derived peptide. The structure and function investigations revealed specific peptide presentation characteristics of Trvu-UB*01:01, which is useful for elucidation of the specific T cell immunity of marsupials and also the immune intervention of emerging viruses (54).

In addition to WPDV, several viruses were identified from the brushtail possum [e.g., possum enterovirus (55), possum adenovirus (56), and possum papillomavirus (57)]. In addition, using an electron microscope, coronavirus and coronavirus-like particles were found from the gastrointestinal contents of brushtail possums (58). However, only WPDV is associated with severe systemic disease characterized by multifocal neurologic disorders (59). WPD has a major impact on the survival and continuation of brushtail possum. The immunological research indicated the immune response in brushtail possum after WPDV infection. In terms of immunohistochemistry, WPDV infection is associated with lymphocytic infiltration in the diseased stroma (brain, liver, and kidney) (16, 59). Subarachnoid infiltration in individuals with mild to severe nonsuppurative encephalitis caused by WPDV infection has a small to moderate number of lymphocytes, but there is no detailed classification of the infiltrating lymphocytes (59, 60). The local function of these infiltrating lymphocytes in the lesion has not been studied in detail.

MHC Is are involved in Ag processing and immune response regulation, and substitution and insertion at seemingly subtle sites in PBG can significantly affect their function (23, 32), including peptide binding and peptide presentation to T cells for TCR recognition. Similar to the insertion of 1 aa existing in the α2 helix of the Trvu-UB*01:01, the insertion has been found in other species. It has been previously reported that the α2 helix is inserted into Leu155 in canine DLA-88*50801 (42). The insertions in the α2 helix of Xenopus laevis Xela-UAA are Glu148 and Val149 (61) and in the green anole lizard Anca-UA*01:01 is Phe152 (62). The median portion of the α2 helix might be a possible insertion hotspot, and the insertion appears to be species specific. Although the presence of the insertion at the α2 helix is not exclusive to marsupials, the 154 insertion of the α2 helix is unique. The previously reported structure of TCR LC13 complexed to HLA-B8/FLRGRAYGL (PDB number 1MI5) showed that CDR1 and CDR3 are indispensable in mediating the interaction between MHC and TCR. The structure of the MHC/peptide complex and TCR visualized that 151–158 residues of the α2 helix interact with the CDR1 loop of TCR (63). In the structure of the ELS4-HLA-B*3501-EPLP (PDB number 2NX5) complex, Arg151 interacted with Tyr48 of the CDR2α loop (64). In the structure of JM22-MP (5866)-HLA-A2 (PDB number 1OGA), H151 in the α2 helix can interact with CDR2 and CDR3 of TCR (63). This may indicate that the insertion site of the α2 helix of Trvu-UB*01:01 might impact the peptide presentation and TCR recognition. The two molecules insert Arg of different conformations in an asymmetric unit of Trvu-UB*01:01, which reflects the possibility of flexibility, possibly because of stacking or solvent exposure resulting in different conformations to recognize different TCRs.

Marsupials are physiologically distinct from bats, but they share some common features of 3-aa insertion. In the present structure, the 3-aa insertion of Trvu-UB*01:01 and bat Ptal-N*01:01 produce some similar characteristics, both of which have the N-terminal extension of the α1 helix, and the conformation change resulted in the protrusion of Asp59 into the PBG. However, the 3-aa insertion of Trvu-UB*01:01 is functionally and structurally different from bat Ptal-N*01:01. Even with the insertion at the same site, Trvu-UB*01:01 still has some conformational differences with bat Ptal-N*01:01; the salient side chain of Asp59 only forms salt bridges with Arg65 without direct interaction with peptide P1-Cys. We conducted P1-C into C1D of peptide WPDV-12 and found that, compared with WPDV-12, there was no increase in either the efficiency of renaturation or the thermal stability of the complex. The 3-aa Ile52Glu53Arg54 insertion only maintains the N-terminal stability of Trvu-UB*01:01.

As one of the key points of the host–pathogen interaction (65, 66), the evolution of the MHC I gene family is closely related to the evolution of the vertebrate immune system (67). The similar insertion characteristics among lower animals and bat MHC I may be a convergent evolution under the pressure of related pathogens (24). Could the insertion of amino acids in MHC I be a common evolutionary response to antiviral infection in lower animals? It has been reported that E3-19K of adenovirus binds to MHC I molecules and retains them in the endoplasmic reticulum, preventing them from being transported to the cell surface (68) and making infected cells less likely to be lysed by specific CTL (69). The crystal structure of E3-19K to human MHC I HLA-A2 showed four interaction sites, of which site 1 is critical, located at the N-terminal of HLA-A2 α1 helix (Fig. 7A). Tyr46 of E3-19K at site 1 can form hydrogen bonds with Glu58 of HLA-A2, hydrogen bonds and salt bridges with Pro57 of HLA-A2, and salt bridges with Gly56 of HLA-A2 (70). Interestingly, the insertion of 3-aa in MHC I of brushtail possum Trvu-UB*01:01 (Fig. 7B, 7C) and bat Ptal-N*01:01 (Fig. 7D, 7E) is precisely at site 1, where HLA-A2 interacts with E3-19K. After superimposing the structure of E3-19K/HLA-A2 with Trvu-UB*01:01, we speculate that the protein may be difficult to bind because of the steric hindrance formed by the extended α helix of 3-aa inserted into the N-terminal of the PBG. Through comparing the MHC I sequence of higher mammals, bats, and lower animals (such as marsupials, monotremes, reptiles, bony fishes, cartilaginous fishes, and amphibians), we found that the insertion is mostly concentrated in the position 52–56 at the α1 helix of MHC I, indicating the hotspot for amino acid insertion (Fig. 7F). If this binding mechanism could be extended to other pathogens, the presence of MHC I molecules inserted at this site might prevent the occurrence of immunosuppression by blocking the binding of certain protein Ags. (Fig. 7G, 7H). However, the specific function and related physiological studies need to be further verified.

FIGURE 7.

Conjecture of the effect of α1 helix insertion on Ag binding. (A) Trvu-UB*01:01 is superimposed with Ptal-N*01:01 (PDB number 6J2D) and Ad2 E3-19K-HLA-A2 (PDB number 4E5X). Trvu-UB*01:01 is indicated in light blue, bat Ptal-N*01:01 is shown in pink, HLA-A2 is displayed in teal, and Ad2 E3-19K is shown in sky-blue. The interaction site 1 between Ad2 E3-19K and HLA-A2 is shown in an orange pear shape. (B) The alignment for Ala49–Glu61 of Trvu-UB*01:01 and Ala49–Tyr59 of HLA-A2. The inserted residues in Trvu-UB*01:01 are displayed in yellow. The color pattern is the same as in (A). (C) Trvu-UB*01:01 is superimposed with HLA-A2, using the same color pattern as in (A), and E3-19K is shown in teal. Ala49–Glu61 of Trvu-UB*01:01 and Ala49–Tyr59 of HLA-A2 are shown in loops; the other areas were treated transparently. A dotted green circle indicates the possible steric hindrance formed by the extended α1 helix and the Y46 residue in E3-19K. (D) The alignment for Ala49–Glu61 of Ptal-N*01:01 and Ala49–Tyr59 of HLA-A2. The inserted residues in Ptal-N*01:01 are displayed in orange. The color pattern is the same as (A). (E) Ptal-N*01:01 is superimposed with HLA-A2, using the same color pattern as in (A), and E3-19K is shown in teal. Ala49–Glu61 of Ptal-N*01:01 and Ala49–Tyr59 of HLA-A2 are shown in loops; the other areas were treated transparently. A dotted green circle indicates the possible steric hindrance formed by the extended α1 helix and the Y46 residue in E3-19K. (F) Sequence alignment of residues at 48 to 61 of the representative MHC I molecules in lower animals (marsupials, monotremes, reptiles, amphibians, and fishes) and bats. The residues marked in red are completely conserved, the insertion sites are marked with cyan rectangles, and the rest of the sequences are marked with purple boxes. The reference sequence for comparison is HLA-A*0201. Icons were made in BioRender (https://biorender.com). (G) Schematic diagram of HLA-A2 combined with E3-19K. (H) Schematic diagram of the effect of steric hindrance from α1-helical amino acids insertion on Ag binding.

FIGURE 7.

Conjecture of the effect of α1 helix insertion on Ag binding. (A) Trvu-UB*01:01 is superimposed with Ptal-N*01:01 (PDB number 6J2D) and Ad2 E3-19K-HLA-A2 (PDB number 4E5X). Trvu-UB*01:01 is indicated in light blue, bat Ptal-N*01:01 is shown in pink, HLA-A2 is displayed in teal, and Ad2 E3-19K is shown in sky-blue. The interaction site 1 between Ad2 E3-19K and HLA-A2 is shown in an orange pear shape. (B) The alignment for Ala49–Glu61 of Trvu-UB*01:01 and Ala49–Tyr59 of HLA-A2. The inserted residues in Trvu-UB*01:01 are displayed in yellow. The color pattern is the same as in (A). (C) Trvu-UB*01:01 is superimposed with HLA-A2, using the same color pattern as in (A), and E3-19K is shown in teal. Ala49–Glu61 of Trvu-UB*01:01 and Ala49–Tyr59 of HLA-A2 are shown in loops; the other areas were treated transparently. A dotted green circle indicates the possible steric hindrance formed by the extended α1 helix and the Y46 residue in E3-19K. (D) The alignment for Ala49–Glu61 of Ptal-N*01:01 and Ala49–Tyr59 of HLA-A2. The inserted residues in Ptal-N*01:01 are displayed in orange. The color pattern is the same as (A). (E) Ptal-N*01:01 is superimposed with HLA-A2, using the same color pattern as in (A), and E3-19K is shown in teal. Ala49–Glu61 of Ptal-N*01:01 and Ala49–Tyr59 of HLA-A2 are shown in loops; the other areas were treated transparently. A dotted green circle indicates the possible steric hindrance formed by the extended α1 helix and the Y46 residue in E3-19K. (F) Sequence alignment of residues at 48 to 61 of the representative MHC I molecules in lower animals (marsupials, monotremes, reptiles, amphibians, and fishes) and bats. The residues marked in red are completely conserved, the insertion sites are marked with cyan rectangles, and the rest of the sequences are marked with purple boxes. The reference sequence for comparison is HLA-A*0201. Icons were made in BioRender (https://biorender.com). (G) Schematic diagram of HLA-A2 combined with E3-19K. (H) Schematic diagram of the effect of steric hindrance from α1-helical amino acids insertion on Ag binding.

Close modal

Among the MHC Is of eutherians, monotremes, reptiles, amphibians, fishes, and marsupials, the insertions in the α1 and α2 helices of the MHC I sequence of lower animals are more frequent. The commonality of insertion or uncommon residues combination will lead to the conformational changes of MHC Is (34). From the formation of hydrogen bond networks to salt bridges, all play a crucial role in stabilizing the structure of MHC I molecules. Furthermore, polymorphisms in insertion sites between different species may be the evolution of antiviral immunity millions of years ago.

In conclusion, through (to our knowledge) the first structural glimpse of marsupial MHC I, we demonstrate the structural similarities between brushtail possum Trvu-UB*01:01 and bat Ptal-N*01:01 and unique features of Trvu-UB*01:01-presenting WPDV-derived peptides raised by unique insertion into the marsupial MHC I. Our results add significantly to the understanding of adaptive immunity of lower mammals. Furthermore, these results may help contribute to the conservation of the protected brushtail possum and other marsupial species.

We thank everyone who assisted in this work.

This work was supported by grants from the National Natural Science Foundation of China (NSFC) (Grant 81971501) and The National Key Research and Development Program (2017YFC1200204). W.J.L. is supported by The National Young Talents Program and the Excellent Young Scientist Program of NSFC (Grant 81822040).

The protein structure presented in this article has been submitted to the Protein Data Bank (https://www.rcsb.org/) under accession number 7EDO.

The online version of this article contains supplemental material.

Abbreviations used in this article

DSC

differential scanning calorimetry

LC-MS/MS

liquid chromatography–tandem MS

β2m

β2m-microglobulin

M1

molecule 1

M2

molecule 2

MHC I

MHC class I

MS

mass spectrometry

PBG

peptide binding groove

PDB

Protein Data Bank

RMSD

root mean square difference

Tm

melting temperature

WPD

wobbly possum disease

WPDV

WPD virus

WT

wild-type

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The authors have no financial conflicts of interest.

Supplementary data