Variable regions A and B in the envelope glycoproteins of feline leukemia virus subgroup B and amphotropic murine leukemia virus interact with discrete receptor domains.

The surface (SU) envelope glycoproteins of feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) are highly related, even in the variable regions VRA and VRB that have been shown to be required for receptor recognition. However, FeLV-B and A-MLV use different sodium-dependent phosphate symporters, Pit1 and Pit2, respectively, as receptors for infection. Pit1 and Pit2 are predicted to have 10 membrane-spanning domains and five extracellular loops. The close relationship of the retroviral envelopes enabled us to generate pseudotype virions carrying chimeric FeLV-B/A-MLV envelope glycoproteins. We found that some of the pseudotype viruses could not use Pit1 or Pit2 proteins but could efficiently utilize specific chimeric Pit1/Pit2 proteins as receptors. By studying Mus dunni tail fibroblasts expressing chimeric Pit1/Pit2 proteins and pseudotype virions carrying chimeric FeLV-B/A-MLV envelopes, we show that FeLV-B and A-MLV VRA and VRB interact in a modular manner with specific receptor domains. Our results suggest that FeLV-B VRA interacts with Pit1 extracellular loops 4 and 5 and that residues Phe-60 and Pro-61 of FeLV-B VRA are essential for receptor choice. However, this interaction is insufficient for infection, and an additional interaction between FeLV-B VRB and Pit1 loop 2 is essential. Similarly, A-MLV infection requires interaction of A-MLV VRA with Pit2 loops 4 and 5 and VRB with Pit2 loop 2, with residues Tyr-60 and Val-61 of A-MLV VRA being critical for receptor recognition. Together, our results suggest that FeLV-B and A-MLV infections require two major discrete interactions between the viral SU envelope glycoproteins and their respective receptors. We propose a common two-step mechanism for interaction between retroviral envelope glycoproteins and cell surface receptors.     

Genetic and biochemical analyses of receptor and cofactor determinants for T-cell-tropic feline leukemia virus infection

Entry by retroviruses is mediated through interactions between the viral envelope glycoprotein and the host cell receptor(s). We recently identified two host cell proteins, FeLIX and Pit1, that are necessary for infection by cytopathic, T-cell-tropic feline leukemia viruses (FeLV-T). Pit1 is a classic multiple transmembrane protein used as a receptor by several other simple retroviruses, including subgroup B FeLV (FeLV-B), and FeLIX is a secreted cellular protein expressed from endogenous FeLV-related sequences (enFeLV). FeLIX is nearly identical to FeLV-B envelope sequences that encode the N-terminal half of the viral surface unit (SU), because these FeLV-B sequences are acquired by recombination with enFeLV. FeLV-B SUs can functionally substitute for FeLIX in mediating FeLV-T infection. Both of these enFeLV-derived cofactors can efficiently facilitate FeLV-T infection only of cells expressing Pit1, not of cells expressing the related transport protein Pit2. We therefore have used chimeric Pit1/Pit2 receptors to map the determinants for cofactor binding and FeLV-T infection. Three distinct determinants appear to be required for cofactor-dependent infection by FeLV-T. We also found that Pit1 sequences within these same domains were required for binding by FeLIX to the Pit receptor. In contrast, these determinants were not all required for receptor binding by the FeLV-B SU cofactors used in this study. These data indicate that cofactor binding is not sufficient for FeLV-T infection and suggest that there may be a direct interaction between FeLV-T and the Pit1 receptor.     

Mutational analysis of the proposed gibbon ape leukemia virus binding site in Pit1 suggests that other regions are important for infection.

Region A of Pit1 (residues 550 to 558 in domain IV) and related receptors has remained the only sequence implicated in gibbon ape leukemia virus (GALV) infection, and an acidic residue at the first position appeared indispensable. The region has also been proposed to be the GALV binding site, but this lacks empirical support. Whether an acidic residue at the first position in this sequence is a definitive requirement for GALV infection has also remained unclear; certain receptors retain function even in the absence of this acidic residue. We report here that in Pit1 an acidic residue is dispensable not only at position 550 but also at 553 alone and at both positions. Further, the virus requires no specific residue at either position. Mutations generated a collection of region A sequences, often with fundamentally different physicochemical properties (overall hydrophobicity or hydrophilicity and net charge of -1, or 0, or +1), and yet Pit1 remained an efficient GALV receptor. A comparison of these sequences and a few previously published ones from highly efficient GALV receptors revealed that every position in region A can vary without affecting GALV entry. Even Pit2 is nonfunctional for GALV only because it has lysine at the first position in its region A, which is otherwise highly diverse from region A of Pit1. We propose that region A itself is not the GALV binding motif and that other sequences are required for virus entry. Indeed, certain Pit1/Pit2 chimeras revealed that sequences outside domain IV are specifically important for GALV infection.     

Specificity in receptor usage by T-cell-tropic feline leukemia viruses: implications for the in vivo tropism of immunodeficiency-inducing variants

Cytopathic, T-cell-tropic feline leukemia viruses (FeLV-T) evolve from FeLV-A in infected animals and demonstrate host cell specificities that are distinct from those of their parent viruses. We recently identified two cellular proteins, FeLIX and Pit1, required for productive infection by these immunodeficiency-inducing FeLV-T variants (M. M. Anderson, A. S. Lauring, C. C. Burns, and J. Overbaugh, Science 287:1828-1830, 2000). FeLV-T is the first example of a naturally occurring type C retrovirus that requires two proteins to gain entry into target cells. FeLIX is an endogenous protein that is highly related to the N-terminal portion of the FeLV envelope protein, which includes the receptor-binding domain. Pit1 is a multiple-transmembrane phosphate transport protein that also functions as a receptor for FeLV-B. The FeLV-B envelope gene is derived by recombination with endogenous FeLV-like sequences, and its product can functionally substitute for FeLIX in facilitating entry through the Pit1 receptor. In the present study, we tested other retrovirus envelope surface units (SUs) with their cognate receptors to determine whether they also could mediate infection by FeLV-T. Cells were engineered to coexpress the transmembrane form of the envelope proteins and their cognate receptors, or SU protein was added as a soluble protein to cells expressing the receptor. Of the FeLV, murine leukemia virus, and gibbon ape leukemia virus envelopes tested, we found that only those with receptor-binding domains derived from endogenous FeLV could render cells permissive for FeLV-T. We also found that there is a strong preference for Pit1 as the transmembrane receptor. Specifically, FeLV-B SUs could efficiently mediate infection of cells expressing the Pit1 receptor but could only inefficiently mediate infection of cells expressing the Pit2 receptor, even though these SUs are able to bind to Pit2. Expression analysis of feline Pit1 and FeLIX suggests that FeLIX is likely the primary determinant of FeLV-T tropism. These results are discussed in terms of current models for retrovirus entry and the interrelationship among FeLV variants that evolve in vivo.     

Reassessing the role of region A in Pit1-mediated viral entry

The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 (region B), that is required for both viral entry and virus binding. Epitope-tagged Pit1 proteins were used to demonstrate that mutations in region B result in improper orientation of Pit1 in the cell membrane. Compensatory mutations in region A can restore proper orientation and full receptor function to these region B mutants. Based on these results, we propose that region A of Pit1 confers competence for viral entry by influencing the topology of the authentic binding site in the membrane and hence its accessibility to a viral envelope protein. Based on glycosylation studies and results obtained by using N- and C-terminal epitope-tagged Pit1, region A and region B mutants, and the transmembrane helices predicted with the PHD PredictProtein algorithm, we propose a new Pit1 topology model.     

Amphotropic murine leukemia virus entry is determined by specific combinations of residues from receptor loops 2 and 4

Pit2 is the human receptor for amphotropic murine leukemia virus (A-MuLV); the related human protein Pit1 does not support A-MuLV entry. Interestingly, chimeric proteins in which either the N-terminal or the C-terminal part of Pit2 was replaced by the Pit1 sequence all retained A-MuLV receptor function. A possible interpretation of these observations is that Pit1 harbors sequences which can specify A-MuLV receptor function when presented in a protein context other than Pit1, e.g., in Pit1-Pit2 hybrids. We reasoned that such Pit1 sequences might be identified if presented in the Neurospora crassa protein Pho-4. This protein is distantly related to Pit1 and Pit2, predicted to have a similar membrane topology with five extracellular loops, and does not support A-MuLV entry. We show here that introduction of the Pit1-specific loop 2 sequence conferred A-MuLV receptor function upon Pho-4. Therefore, we conclude that (i) a functional A-MuLV receptor can be constructed by combining sequences from two proteins each lacking A-MuLV receptor function and that (ii) a Pit1 sequence can specify A-MuLV receptor function when presented in another protein context than that provided by Pit1 itself. Previous results indicated a role of loop 4 residues in A-MuLV entry, and the presence of a Pit2-specific loop 4 sequence was found here to confer A-MuLV receptor function upon Pho-4. Moreover, the introduction of a Pit1-specific loop 4 sequence, but not of a Pit2-specific loop 4 sequence, abolished the A-MuLV receptor function of a Pho-4 chimera harboring the Pit1-specific loop 2 sequence. Together, these data suggest that residues in both loop 2 and loop 4 play a role in A-MuLV receptor function. A-MuLV is, however, not dependent on the specific Pit2 loop 2 and Pit2 loop 4 sequences for entry; rather, the role played by loops 2 and 4 in A-MuLV entry can be fulfilled by several different combinations of loop 2 and loop 4 sequences. We predict that the residues in loops 2 and 4, identified in this study as specifying A-MuLV receptor function, are to be found among those not conserved among Pho-4, Pit1, and Pit2.     

Global analysis of small molecule binding to related protein targets

We report on the integration of pharmacological data and homology information for a large scale analysis of small molecule binding to related targets. Differences in small molecule binding have been assessed for curated pairs of human to rat orthologs and also for recently diverged human paralogs. Our analysis shows that in general, small molecule binding is conserved for pairs of human to rat orthologs. Using statistical tests, we identified a small number of cases where small molecule binding is different between human and rat, some of which had previously been reported in the literature. Knowledge of species specific pharmacology can be advantageous for drug discovery, where rats are frequently used as a model system. For human paralogs, we demonstrate a global correlation between sequence identity and the binding of small molecules with equivalent affinity. Our findings provide an initial general model relating small molecule binding and sequence divergence, containing the foundations for a general model to anticipate and predict within-target-family selectivity.     

Mapping the HSP90 binding region of the glucocorticoid receptor

In animal cells, unliganded steroid receptors are complexed with a 90-kDa heat shock protein, HSP90; hormone binding by the receptor leads to the release of HSP90. We found that the 795-amino acid rat glucocorticoid receptor protein formed oligomeric complexes in vitro upon synthesis in rabbit reticulocyte lysates; these oligomers also dissociated in the presence of hormone. Similar complexes formed when X795, a receptor derivative containing only the C-terminal half (amino acids 407-795) of the protein, was translated in vitro. Moreover, X795 was co-immunoadsorbed from the reticulocyte lysates together with HSP90 by three different anti-HSP90 monoclonal antibodies, indicating that the in vitro translated receptor binds HSP90 and that the interaction occurs within the C-terminal half of the receptor. To localize the HSP90 binding region in greater detail, various deletion mutants of X795 were translated in vitro and assayed for oligomer formation and for co-immunoadsorption with HSP90. The results indicated that HSP90 interacted with the receptor within a subregion of the hormone binding domain, between amino acids 568 and 616. These findings are consistent with the proposal that HSP90 may participate in the mechanism of signal transduction by steroid receptors.     

Control of estrogen receptor ligand binding by Hsp90

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.     

Substrate adaptabilities of Thermotogae mannan binding proteins as a function of their evolutionary histories

The Thermotogae possess a large number of ATP-binding cassette (ABC) transporters, including two mannan binding proteins, ManD and CelE (previously called ManE). We show that a gene encoding an ancestor of these was acquired by the Thermotogae from the archaea followed by gene duplication. To address the functional evolution of these proteins as a consequence of their evolutionary histories, we measured the binding affinities of ManD and CelE orthologs from representative Thermotogae. Both proteins bind cellobiose, cellotriose, cellotetraose, β-1,4-mannotriose, and β-1,4-mannotetraose. The CelE orthologs additionally bind β-1,4-mannobiose, laminaribiose, laminaritriose and sophorose while the ManD orthologs additionally only weakly bind β-1,4-mannobiose. The CelE orthologs have higher unfolding temperatures than the ManD orthologs. An examination of codon sites under positive selection revealed that many of these encode residues located near or in the binding site, suggesting that the proteins experienced selective pressures in regions that might have changed their functions. The gene arrangement, phylogeny, binding properties, and putative regulatory networks suggest that the ancestral mannan binding protein was a CelE ortholog which gave rise to the ManD orthologs. This study provides a window on how one class of proteins adapted to new functions and temperatures to fit the physiologies of their new hosts.     

Identification of Envelope Determinants of Feline Leukemia Virus Subgroup B That Permit Infection and Gene Transfer to Cells Expressing Human Pit1 or Pit2

The retroviral vector systems that are in common use for gene therapy are designed to infect cells expressing either of two widely expressed phosphate transporter proteins, Pit1 or Pit2. Subgroup B feline leukemia viruses (FeLV-Bs) use the gibbon ape leukemia virus receptor, Pit1, as a receptor for entry. Our previous studies showed that some chimeric envelope proteins encoding portions of FeLV-B could also enter cells by using a related receptor protein, Pit2, which serves as the amphotropic murine leukemia virus receptor (S. Boomer, M. Eiden, C. C. Burns, and J. Overbaugh, J. Virol. 71:8116--8123, 1997). Here we show that an arginine at position 73 within variable region A (VRA) of the FeLV-B envelope surface unit (SU) is necessary for viral entry into cells via the human Pit2 receptor. However, C-terminal SU sequences have a dominant effect in determining human Pit2 entry, even though this portion of the protein is outside known receptor binding domains. This suggests that a combination of specific VRA sequences and C-terminal sequences may influence interactions between FeLV-B SU and the human Pit2 receptor. Binding studies suggest that the C-terminal sequences may affect a postbinding step in viral entry via the Pit2 receptor, although in all cases, binding of FeLV-B SU to human Pit2 was weak. In contrast, neither the arginine 73 nor specific C-terminal sequences are required for efficient binding or infection with Pit1. Taken together, these data suggest that different residues in SU may interact with these two receptors. The specific FeLV-Bs described here, which can enter cells using either human Pit receptor, may be useful as envelope pseudotypes for viruses used in gene therapy.     

Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains

Viral spillover from animal reservoirs can trigger public health crises and cripple the world economy. Knowing which viruses are primed for zoonotic transmission can focus surveillance efforts and mitigation strategies for future pandemics. Successful engagement of receptor protein orthologs is necessary during cross-species transmission. The clade 1 sarbecoviruses including Severe Acute Respiratory Syndrome-related Coronavirus (SARS-CoV) and SARS-CoV-2 enter cells via engagement of angiotensin converting enzyme-2 (ACE2), while the receptor for clade 2 and clade 3 remains largely uncharacterized. We developed a mixed cell pseudotyped virus infection assay to determine whether various clades 2 and 3 sarbecovirus spike proteins can enter HEK 293T cells expressing human or Rhinolophus horseshoe bat ACE2 proteins. The receptor binding domains from BtKY72 and Khosta-2 used human ACE2 for entry, while BtKY72 and Khosta-1 exhibited widespread use of diverse rhinolophid ACE2s. A lysine at ACE2 position 31 appeared to be a major determinant of the inability of these RBDs to use a certain ACE2 sequence. The ACE2 protein from Rhinolophus alcyone engaged all known clade 3 and clade 1 receptor binding domains. We observed little use of Rhinolophus ACE2 orthologs by the clade 2 viruses, supporting the likely use of a separate, unknown receptor. Our results suggest that clade 3 sarbecoviruses from Africa and Europe use Rhinolophus ACE2 for entry, and their spike proteins appear primed to contribute to zoonosis under the right conditions.

Knowing which viruses are primed for zoonotic transmission can focus surveillance efforts and mitigation strategies for future pandemics. This study shows that SARS-like coronaviruses identified in bats from Europe and Africa can use a range of horseshoe bat ACE2s for entry. In addition, viruses found in Russia and Kenya also have the ability to at least weakly use human ACE2.     

Identification of Xenopus laevis sperm and egg envelope binding components on nitrocellulose membranes

Interacting egg envelope and sperm surface components were identified for Xenopus laevis using blotting methods. Sperm were extracted with sodium dodecyl sulfate (SDS), the extracted proteins separated by gel electrophoresis and blotted, and the blots treated with 125I-labeled heat solubilized envelopes. The converse experiment was also performed where envelope components were separated by gel electrophoresis, blotted, and the blots treated with 125I-labeled sperm components. Blotted sperm components with apparent molecular weights of 14K, 19K, 25K, and 35K selectively bound the solubilized envelopes. All of the envelope binding components were found to be localized on the sperm surface by radioiodinating intact sperm using Iodo-Gen. The blotted egg envelope component with an apparent molecular weight of 37K selectively bound to solubilized sperm components, and this binding was due to the protein moiety of the glycoprotein. 125I-labeled heat solubilized envelopes from unfertilized and fertilized eggs showed the same pattern of binding to blotted sperm components. Selected sulfated carbohydrates (fucoidan, dextran sulfate, and heparin, but not chondroitin sulfate) inhibited fertilization and binding of 125I-labeled heat solubilized envelopes to blotted sperm extract. Thus, the binding of heat solubilized envelopes to electrophoretically separated and blotted sperm proteins may reflect cellular interactions.     

Enhanced recA protein binding to Z DNA represents a kinetic perturbation of a general duplex DNA binding pathway

recA protein binding to duplex DNA is enhanced when a B form DNA substrate is replaced with a left-handed Z form helix. This represents a kinetic rather than an equilibrium effect. Binding to Z DNA is much faster than binding to B DNA. In other respects, binding to the two DNA forms is quite similar. recA protein binds to B or Z DNA with a stoichiometry of 1 monomer/4 base pairs. The final protein filament exhibits a right-handed helical structure when either B or Z form DNAs are bound. There are only two evident differences: the kcat for ATP hydrolysis is reduced 3-4-fold when Z DNA is bound, and recA binding at equilibrium is less stable on Z DNA than on B DNA. At steady state, the binding favors B DNA in competition experiments. The results indicate that Z DNA binding by recA protein follows the same pathway as for recA binding to B DNA, but that the nucleation step is faster on the Z form helix.     

Receptor co-operation in retrovirus entry: recruitment of an auxiliary entry mechanism after retargeted binding.

We have constructed Moloney murine leukemia virus (MoMLV)-derived envelope glycoproteins (AMO) displaying an amino-terminal Ram-1-binding domain in which a variety of different amino acid spacers have been inserted between the displayed domain and the MoMLV surface (SU) subunit. Titres of retroviruses generated with these chimeric envelopes were enhanced on cells expressing both Ram-1 and Rec-1 receptors compared with the titres on cells expressing only one or other receptor type. The absolute viral titres and the degree of titre enhancement due to receptor cooperativity were highly variable between the different chimeric envelopes and were determined primarily by the properties of the interdomain spacer. An extreme example of receptor co-operativity was encountered when testing Ram-1-targeted AMOPRO envelopes with specific proline-rich interdomain spacers. AMOPRO viruses could not enter cells expressing only Rec-1 or only Ram-1 but could efficiently infect cells co-expressing both receptors. The data are consistent with a model for receptor co-operativity in which binding to the targeted (Ram-1) receptor triggers conformational rearrangements of the envelope that lead to complete unmasking of the hidden Rec-1-binding domain, thereby facilitating its interaction with the viral (Rec-1) receptor which leads to optimal fusion triggering.     

Mapping of equine lentivirus receptor 1 residues critical for equine infectious anemia virus envelope binding

The equine lentivirus receptor 1 (ELR1), a member of the tumor necrosis factor receptor (TNFR) protein family, has been identified as a functional receptor for equine infectious anemia virus (EIAV). Toward defining the functional interactions between the EIAV SU protein (gp90) and its ELR1 receptor, we mapped the gp90 binding domain of ELR1 by a combination of binding and functional assays using the EIAV SU gp90 protein and various chimeric receptor proteins derived from exchanges between the functional ELR1 and the nonbinding homolog, mouse herpesvirus entry mediator (murine HveA). Complementary exchanges of the respective cysteine-rich domains (CRD) between the ELR1 and murine HveA proteins revealed CRD1 as the predominant determinant of functional gp90 binding to ELR1 and also to a chimeric murine HveA protein expressed on the surface of transfected Cf2Th cells. Mutations of individual amino acids in the CRD1 segment of ELR1 and murine HveA indicated the Leu70 in CRD1 as essential for functional binding of EIAV gp90 and for virus infection of transduced Cf2Th cells. The specificity of the EIAV SU binding domain identified for the ELR1 receptor is fundamentally identical to that reported previously for functional binding of feline immunodeficiency virus SU to its coreceptor CD134, another TNFR protein. These results indicate unexpected common features of the specific mechanisms by which diverse lentiviruses can employ TNFR proteins as functional receptors.     

Fungal phosphate transporter serves as a receptor backbone for gibbon ape leukemia virus.

Pit1, the receptor for gibbon ape leukemia virus (GALV), is proposed to be an integral membrane protein with five extracellular loops. Chimeras made between Pit1 homologs differing in permissivity for infection and between Pit1 and the related protein Pit2 have shown that the fourth extracellular loop plays a critical role in infection. However, further elucidation of the roles of the extracellular loops in infection is hampered by the high level of sequence similarity among these proteins. The sodium-dependent phosphate transporter, Pho-4, from the filamentous fungus Neurospora crassa is distantly related to Pit1 and -2, showing an amino acid identity of only 35% to Pit1 in the putative extracellular loops. We show here that Pho-4 itself does not function as a receptor for GALV. Introduction of 12 Pit1-specific amino acid residues in the putative fourth extracellular loop of Pho-4 resulted in a functional GALV receptor. Therefore, the presence of a Pit1 loop 4-specific sequence is sufficient to confer receptor function for the mammalian retrovirus GALV on the fungal phosphate transporter Pho-4.     

A computational assessment of the predicted structures of Human Macrophage Migration Inhibitory Factor 1 orthologs in parasites and its affinity to human CD74 receptor

The human macrophage migration inhibitory factor 1 (Hu‐MIF‐1) is a protein involved in the inflammatory and immunology response to parasite infection. In the present study, the existence of Hu‐MIF‐1 from parasites have been explored by mining WormBase. A total of 35 helminths were found to have Hu‐MIF‐1 homologs, including some parasites of importance for public health. Physicochemical, structural, and biological properties of Hu‐MIF‐1 were compared with its orthologs in parasites showing that most of these are secretory proteins, with positive net charge and presence of the Cys‐Xaa‐Xaa‐Cys motif that is critical for its oxidoreductase activity. The inhibitor‐binding site present in Hu‐MIF‐1 is well conserved among parasite MIFs suggesting that Hu‐MIF inhibitors may target orthologs in pathogens. The binding of Hu‐MIF‐1 to its cognate receptor CD74 was predicted by computer‐assisted docking, and it resulted to be very similar to the predicted complexes formed by parasite MIFs and human CD74. More than 1 plausible conformation of MIFs in the extracellular loops of CD74 may be possible as demonstrated by the different predicted conformations of MIF orthologs in complex with CD74. Parasite MIFs in complex with CD74 resulted with some charged residues oriented to CD74, which was not observed in the Hu‐MIF‐1/CD74 complex. Our findings predict the binding mode of Hu‐MIF‐1 and orthologs with CD74, which can assist in the design of novel MIF inhibitors. Whether the parasite MIFs function specifically subvert host immune responses to suit the parasite is an open question that needs to be further investigated. Future research should lead to a better understanding of parasite MIF action in the parasite biology.