Secondary structure composition and pH-dependent conformational changes of soluble recombinant HLA-DM.
HLA-DM catalyzes the release of invariant chain fragments from newly synthesized major histocompatibility complex (MHC) class II molecules, stabilizes empty class II molecules, and edits class II-associated peptides by preferentially releasing those that are loosely bound. The ability of HLA-DM to carry out these functions in vitro is pH dependent, with an optimum at pH 4.5-5.5 and poor activity at pH 7. The structural basis for these properties of HLA-DM is unknown. Sequence homology suggests that HLA-DM resembles classical, peptide-binding MHC class II molecules. In this study, we examined whether HLA-DM has a secondary structure composition consistent with an MHC fold and whether HLA-DM changes conformation between pH 5 and pH 7. Far-UV circular dichroism (CD) spectra of recombinant soluble HLA-DM (sDM) indicate that HLA-DM belongs to the alpha/beta class of proteins and structurally resembles both MHC class I and class II molecules. The CD peak around 198 nm increases upon going from neutral to endosomal pH and drops sharply upon denaturation below pH 3.5, distinguishing at least three states of sDM: the denatured state and two highly similar folded states. Fluorescence emission spectra show a slight blue-shift and a approximately 20% drop in intensity at pH 5 compared with pH 7. Unfolding experiments using guanidinium chloride show that the stability of sDM is somewhat reduced but not lost at pH 5. These results indicate that sDM undergoes a pH-dependent conformational change between neutral and endosomal pH. The change seems to involve both hydrogen bonding patterns and the hydrophobic core of sDM and may contribute to the pH dependence of DM activity.
Department of Pediatrics, Stanford University, Stanford, California 94305, USA. rbushch@leland.stanford.edu
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R
Busch
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Reich
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Zaller
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Sloan
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Mellins
D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D006681:HLA-D Antigens / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D000949:Histocompatibility Antigens Class II; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry
C416189:H2-M antigens; D006681:HLA-D Antigens; C084245:HLA-DM antigens; D000949:Histocompatibility Antigens Class II; D011994:Recombinant Proteins; D014364:Tryptophan; D019791:Guanidine