Influence of inflammation-related changes on conformational characteristics of HLA-B27 subtypes as detected by IR spectroscopy Heinz Fabian1, Bernhard Loll2, Hans Huser3, Dieter Naumann1, Barbara Uchanska-Ziegler3 and Andreas Ziegler3 Robert Koch-Institut, Berlin, Germany Institut fur Chemie und Biochemie, Abteilung Strukturbiochemie, Freie Universitat Berlin, Germany ă ă Institut fur Immungenetik, Charite-Universita ătsmedizin Berlin, Freie Universitat Berlin, Germany ă ¨ Keywords ankylosing spondylitis; citrullination; conformational differences; HLA-B27 subtypes; IR spectroscopy Correspondence D Naumann, Robert Koch-Institut, P 25, Nordufer 20, D-13353 Berlin, Germany Fax: +49 30 1875 42606 Tel: +49 30 1875 42259 E-mail: naumannd@rki.de (Received December 2010, revised March 2011, accepted 11 March 2011) doi:10.1111/j.1742-4658.2011.08097.x Inflammatory processes are accompanied by the post-translational modification of certain arginine residues to yield citrulline, and a pH decrease in the affected tissue, which might influence the protonation of histidine residues within proteins We employed isotope-edited IR spectroscopy to investigate whether conformational features of two human major histocompatibility antigen class I subtypes, HLA-B*2705 and HLA-B*2709, are affected by these changes Both differ only in residue 116 (Asp vs His) within the peptide-binding grooves, but are differentially associated with inflammatory rheumatic disorders Our analyses of the two HLA-B27 subtypes in complex with a modified self-peptide containing a citrulline RRKWURWHL (U = citrulline) revealed that the heavy chain is more flexible in the HLA-B*2705 subtype than in the HLA-B*2709 subtype Together with our previous studies of HLA-B27 subtypes complexed with the unmodified self-peptide RRKWRRWHL, these findings support the existence of subtype-specific conformational features of the heavy chains under physiological conditions, which are undetectable by X-ray crystallography and exist irrespective of the sequence of the bound peptide and its binding mode They might thus influence antigenic properties of the respective HLA-B27 subtype Furthermore, a decrease in the pH from 7.5 to 5.6 during the analyses had an influence only on HLA-B*2709 complexed with the unmodified self-peptide, where His116 is not contacted by any peptide side chain This permits us to conclude that histidines, and in particular His116, influence the stability of MHC:peptide complexes The conditions prevailing in inflammatory environments in vivo might thus also exert an impact on selected conformational features of HLA-B27:peptide complexes Structured digital abstract l B*27 and VIPR bind by biophysical (View interaction) Abbreviations AS, ankylosing spondylitis; H ⁄ D, hydrogen ⁄ deuterium; HC, heavy chain; HLA, human leukocyte antigen; b2m, b2-microglobulin; MHC, major histocompatibility complex; pLMP2, (RRRWRRLTV); pVIPR, (RRKWRRWHL); pVIPR-U5, (RRKWURWHL; U = citrulline); TIS, (RRLPIFSRL) FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1713 Influence of inflammatory environment on HLA-B27 H Fabian et al Introduction Major histocompatibility complex (MHC) class I molecules are cell-surface membrane glycoproteins that consist of a highly polymorphic heavy chain (HC) noncovalently associated with a light chain, b2-microglobulin (b2m), and a peptide derived from intracellular proteins [1] Recognition of these peptide-loaded MHC molecules by cellular ligands on effector cells triggers immune responses [2] For human leukocyte antigen (HLA) class I molecules, peptides derived from self- or nonself proteins are usually 8–12 amino acids long and are accommodated in a binding groove of the molecule by means of HLA allele-characteristic ‘anchor’ amino acids This selectivity of MHC molecules towards certain anchor residues of peptides provides the basis for HLA subtype-specific immune responses and impacts on disease associations as described, for example, for the group of HLA-B27 alleles [3–6] It is known that the citrullination of proteins, a post-translational modification, influences immune responses and inflammatory reactions [7,8] This modification involves arginine, which is strongly basic, whereas the resulting citrulline is a neutral amino acid [9] Citrullination is found within synovial tissue from patients with reactive arthritis, an HLA-B27-associated disorder [10] Moreover, fragments derived from citrullinated polypeptides are most likely also available for presentation by MHC antigens in patients with ankylosing spondylitis (AS), another spondylarthropathy that is even more strongly associated with HLA-B27 than reactive arthritis [2,11] Although distinct, both diseases are also characterized by inflammatory processes that are, by their very nature, associated with a decrease in the pH value of the affected tissues [12], leading to proton concentrations that can be elevated greatly (100- to 200-fold) The pH decrease is expected to affect primarily histidine residues, due to their sensitivity to relatively small pH shifts at physiologically relevant values (the pKa of histidine in proteins is  6.6) [13] It is well established that protonation of ionizable groups in folded proteins may contribute to their conformational stability [14] Among the HLA-B27 subtypes, HLA-B*2705 (in short, B*2705) is strongly associated with AS, whereas another, HLA-B*2709 (in short, B*2709) is not [2,3,11] The proteins encoded by these two alleles differ only by a micropolymorphism (Asp116 in B*2705 and His116 in B*2709) within the peptide-binding groove formed by each of the HC [1,3] Detailed functional, structural and thermodynamic studies of these very closely related subtypes have been carried out to shed light on the molecular mechanisms underlying their differential 1714 association with AS [15–26] High-resolution crystal structures of these subtypes complexed with peptides constituting the HLA-B27 repertoire reveal that some peptides, such as the self-peptides TIS (RRLPIFSRL) or pCatA (IRAAPPPLF), are displayed very similarly by the two HLA-B27 subtypes [21,26], whereas the viral peptide pLMP2 (RRRWRRLTV) [22] and the self-peptide pVIPR (RRKWRRWHL) [18] exhibit drastically different conformations In addition, pVIPR is bound in a canonical single conformation by B*2709, but in an exceptional dual conformation by the AS-associated B*2705 subtype One of the conformations observed in B*2705:pVIPR is identical to that seen in B*2709, whereas in the other, peptide Arg5 (pArg5) forms a salt bridge to HC residue Asp116, resulting in a noncanonical binding mode of the ligand The dual conformation of this ligand in B*2705 has also been linked to differential T-cell responses among the two HLA-B27 subtypes [15,18] Recently, an isotope-edited IR spectroscopic study of B*2709:pVIPR and B*2705:pVIPR demonstrated that the HC is more flexible in the B*2705 subtype than in the B*2709 subtype at physiological temperatures [27] Furthermore, similar conformational differences between the HC of the two subtypes were also found in complexes with the viral peptide pLMP2 and the self-peptide TIS [28] Collectively, these findings reveal the existence of subtype-specific, but peptide sequence- and conformation-independent conformational differences between the two HLA-B27 HC at physiological temperatures, which to date have not been detectable using X-ray crystallography To approach the situation prevailing in inflamed tissue, we have now extended these IR spectroscopic studies to a citrullinated self-peptide, pVIPR-U5 (RRKWURWHL; U = citrulline) and performed experiments at a lower pH value This peptide is presented by the two HLA-B27 molecules in binding modes that differ drastically not only from each other [29], but also from the conformations exhibited by the noncitrullinated version of the peptide [18] Specifically, pVIPR-U5 is displayed by B*2705 in a canonical conformation (Fig 1A), but exhibits a noncanonical binding mode in the B*2709 subtype, where the side chain of citrulline at peptide position (pU5) is embedded within the binding groove and forms a hydrogen bond to His116 of the HC (Fig 1B) The comparative IR spectroscopic analyses described here address the question of whether the previously observed difference in flexibility between the B*2705 and B*2709 HC is also found when a peptide assumes, FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS H Fabian et al Fig Structure of the pVIPR-U5 peptide in complex with B*2705 and B*2709 The peptide is depicted from the side of the a2 helix (not shown) The floor of the peptide-binding groove and the a1 helix are shown in grey ribbon representation, the subtype-specific residue 116 (Asp116 or His116) is indicated in green (A) The pVIPR-U5 peptide is drawn as a purple stick model bound to B*2705 (B) The pVIPR-U5 peptide is drawn as a yellow stick model anchored to B*2709 by a hydrogen bond connecting pU5O7 and His116NE2, as indicated by a dashed red line Oxygen atoms are shown in red, nitrogen atoms in blue B*2705 binds the peptide in canonical conformation, while it is presented by B*2709 in a noncanonical binding mode because of citrullination, a noncanonical binding mode in the B*2709 subtype Furthermore, we investigated whether the micropolymorphism that distinguishes the two HLA-B27 subtypes exerts an influence on the stability of the complexes when the pH is lowered to a value representative of an inflammatory environment Results Infrared absorbance spectra of HLA-B27:pVIPR-U5 complexes The IR spectroscopic behaviour of the B*2709 ⁄ 13 C-b2m:pVIPR-U5 and B*2705 ⁄ 13C-b2m:pVIPR-U5 Influence of inflammatory environment on HLA-B27 complexes was initially studied at pH 7.5 after transfer into D2O-buffer (Fig 2A) The use of 13C-labelled b2m for reconstitution with separately expressed unlabelled HC in the presence of the corresponding peptide greatly reduced overlapping of its amide I band with that of the HC in the spectroscopic analyses The spectra of the two HLA-B27:pVIPR-U5 complexes were very similar to those previously determined for complexes with the unmodified self-peptides pVIPR [27], TIS [28] or the viral peptide pLMP2 [28], in a given subtype A feature at  1594 cm)1 due to 13C-labelled b2m and a dominant broad absorbance corresponding to the HC centred at  1640 cm)1 were observed The underlying HC-specific band components between 1620 and 1700 cm)1 had previously been assigned to different secondary structure elements of the HC Bands at  1650 and 1643 cm)1 are primarily due to helical structures which change as a consequence of hydrogen ⁄ deuterium (H ⁄ D) exchange, whereas a strong band component at  1624 cm)1 and weaker band components between 1693 and 1681 cm)1 are due to the b sheets of the HC [27] Because 38% and 23% of the protein backbone of the HC is formed by b sheet and a-helical structures, respectively, spectral components attributed to these structures dominate the IR spectrum The spectral contributions of the pVIPR-U5 nonapeptide are ‘buried’ under those of the 276 HC residues The same holds true for the spectral features associated with the His fi Asp exchange in the HC The amino acid side chain of Asp gives rise to a relatively strong absorption band between 1550 and 1585 cm)1, which overlaps with an absorption band due to Glu of similar intensity [30,31] Taking into account the 34 Asp and Glu residues in B*2709, the spectral contribution of one additional Asp in B*2705 is practically negligible This can be expected even more for His, because its side-chain absorption band around 1600 cm)1 is very weak [31] The remaining IR intensity at  1545 cm)1 (amide N–H deformation vibration) h after transfer into D2O buffer, together with the presence of a band at  3286 cm)1 (N–H stretching vibration), shows that a number of amide NH groups of the HC in the two complexes are protected from H ⁄ D exchange The band at  3286 cm)1 (amide A) is the best indicator of residual nonexchanged N-H groups, owing to the lack of other protein absorption in the range 3200– 3400 cm)1 [30] Unfortunately, the very strong water absorption band at  3400 cm)1 (O–H stretching vibration) prevents one from obtaining the amide A band of the HLA-B27:peptide complex in H2O buffer, even when using IR transmission cells of only a few lm pathlength Thus, the amount of nonexchanged FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1715 H Fabian et al F E 1514 1592 1445 1562 1542 1560 3286 100 mA 3400 3300 3200 1700 Wavenumber (cm–1) 1600 1500 1448 1545 1515 1594 1640 1651 1624 1598 1688 20 mA 1694 1688 A 3292 ΔA × B Absorbance C 1651 1624 1650 1624 3314 3306 3281 1693 1691 D 3293 2nd derivative × 100 Δ2nd derivative × 100 Influence of inflammatory environment on HLA-B27 1400 Fig HLA-B27 complexes measured in D2O buffer at pH 7.5 (Lower) (A) IR absorbance spectra of B*2709 ⁄ 13C-b2m:pVIPR-U5 (black trace) and of B*2705 ⁄ 13C-b2m:pVIPR-U5 (red trace), both measured at 15 °C h after transfer into D2O buffer The spectra of the two samples were normalized using the tyrosine absorption band at 1514 cm)1 as an internal intensity standard (Middle) Differences of IR spectra of the HLA-B27:peptide complexes, all measured at 15 °C h after transfer into D2O-buffer (B) B*2709 ⁄ 13C-b2m:pVIPR-U5 ) B*2705 ⁄ 13Cb2m:pVIPR-U5 and (C) B*2709 ⁄ 13C-b2m:pVIPR ) B*2705 ⁄ 13C-b2m:pVIPR The IR data of B*2709 ⁄ 13C-b2m:pVIPR and B*2705 ⁄ 13Cb2m:pVIPR are from previous work by our group [27] Note that the absorbance scale for the difference spectra (B, C) was expanded by a factor of five compared with the scale of the absorbance spectra (A) (Upper) (D) Second derivatives of the IR spectra of B*2709 ⁄ 13Cb2m:pVIPR-U5 (black trace) and B*2705 ⁄ 13C-b2m:pVIPR-U5 (red trace) at 15 °C (E) IR difference spectra (B*2709 ⁄ 13 C-b2m:pVIPR-U5 ) B*2705 ⁄ 13C-b2m:pVIPR-U5) of the second derivatives at 15 °C (black trace) and at 90 °C (red trace) (F) IR difference spectra of the second derivatives of experiments with two independent preparations of each HLA-B27:pVIPR-U5 complex (black trace; B*2709; red trace, B*2705), demonstrating the high reproducibility of the experimental data amide protons for the partially exchanged state of the complex at the beginning of the experiment in D2O was approximated (Fig S1) by setting the difference in peak intensity of amide II at 1550 cm)1 between the spectra of the sample in H2O buffer (0% exchange) and after thermal denaturation of the complex at 90 °C (fully deuterated state) to 100% This approach is only an approximation because: (a) the amide II bands of different conformations of proteins have 1716 different peak maxima, and (b) IR bands due to amino acid side-chain absorptions (overlapping bands of Glu and Asp) between 1550 and 1585 cm)1 may overlap with residual amide II band features and may change as a function of conformational changes Keeping this in mind, the IR data suggest that  50% of the amide protons of the B*2709 ⁄ 13C-b2m:pVIPR-U5 complex remained unexchanged h after transfer into D2O buffer FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS H Fabian et al Detection of HLA-B27 subtype-dependent conformational properties The IR difference spectrum obtained by subtracting the spectrum of B*2705 ⁄ 13C-b2m:pVIPR-U5 from that of B*2709 ⁄ 13C-b2m:pVIPR-U5 at 15 °C (Fig 2B) is characterized by spectral features in the amide A, amide I¢, and amide II ⁄ II¢ region The positive IR difference band at  3292 cm)1 demonstrates the presence of less H ⁄ D-exchanged amide groups in the HC of B*2709 compared with the B*2705 subtype, which is supported by the broad positive difference feature at  1560 cm)1 Because less H ⁄ D exchange means less flexibility of the proteins’ core regions to make them accessible to the solvent, the IR data demonstrate that the B*2705 HC is more flexible than the B*2709 HC The difference feature at  3292 cm)1 (Fig 2B) accounts for only  2% of the total area under the amide A band of the B*2709 ⁄ 13C-b2m:pVIPR-U5 complex (black trace in Fig 2A) relative to a baseline between 3180 and 3410 cm)1 Taking the estimated 50% of nonexchanged amide protons of the B*2709 ⁄ 13C-b2m:pVIPR-U5 sample as reference, the IR data indicate that the observed differences in flexibility between the two subtypes might be restricted to only some residues of their HC The difference features around 1688 and 1624 cm)1 are due to spectral characteristics associated with amide I¢ band components attributed to the b sheets of the HC, suggesting fine differences in the hydrogenbonding pattern of the b-type structures of the HC in the two HLA-B27 samples By contrast, no positive or negative features are observed around 1594 cm)1 (Fig 2B), indicating identical peak positions of the bsheet band of 13C-labelled b2m in the two samples In turn, these characteristics suggest a very similar degree of H ⁄ D exchange of the amide protons of b2m in B*2705 ⁄ 13C-b2m:pVIPR-U5 when compared with that in B*2709 Remarkably, the spectral differences observed herein for the two complexes with pVIPR-U5 (Fig 2B) revealed difference bands very similar to those found previously by us in case of the pVIPRcomplexed B*2709 and B*2705 subtypes (Fig 2C), including a positive difference feature in the amide A region and positive and negative features in the amide I¢ ⁄ II region of the spectrum Moreover, the IR difference spectroscopic features (B*2709 ⁄ 13Cb2m:pVIPR-U5 ) B*2705 ⁄ 13C-b2m:pVIPR-U5) did not change appreciably between 15 and 55 °C (data not shown), indicating that the conformational differences between the two HLA-B27 complexes persist over this temperature range, as observed formerly for the two HLA-B27:pVIPR complexes [27] Influence of inflammatory environment on HLA-B27 To estimate the number and position of individual components under the broad amide I ⁄ I¢ band contours, we also employed derivative spectroscopy This method allows to visualize fine differences in the position, intensity and shape of band components in greater detail than by making simple comparisons of the original spectra [31,32] Differences in peak position and ⁄ or intensities of the amide I¢ bands attributed to the b sheets of the HC are clearly visible by comparing the second derivatives of the spectra of the two HLA-B27:pVIPR-U5 complexes collected at 15 °C and pH 7.5 (black and red traces in Fig 2D) Striking is the difference in peak position of the amide I¢ band due to the high-frequency b-sheet band component at 1691 ⁄ 93 cm)1 which gives rise to obvious positive and negative features by subtracting the second derivative IR spectrum of B*2705:pVIPR-U5 from that of B*2709:pVIPR-U5 (Fig 2E) These differences, together with the minor spectral differences of the lowfrequency b-sheet component at  1624 cm)1, indicate the presence of fine differences in the relative orientation of the b strands of the HC in the two HLA-B27 samples at physiological temperatures The features between 1635 and 1655 cm)1 (Fig 2E) suggest a more intense and ⁄ or slightly shifted amide I¢ band component in the spectrum of B*2709 compared with that of B*2705 Subtle structural differences between the HC of the two HLA-B27 subtypes, which cannot be specified at present, are also indicated by the amide A band components at 3306 and 3314 cm)1 of B*2709 and B*2705, respectively (Fig 2D,E) The high-temperature IR difference spectrum is featureless (red trace in Fig 2E), indicating the loss of all conformational differences between the two HLA-B27 subtypes Moreover, the subtype-dependent spectral differences (black trace in Fig 2E) were much more pronounced than the spectral differences between two independent preparations of the corresponding complexes (Fig 2F) This provides evidence that the observed spectral differences at low temperatures are really significant, and demonstrates the high quality of the experimental data (also see [28]) Subtype-dependent conformational properties as deduced from IR spectroscopy in water IR measurements in D2O provide valuable information on both the structure and flexibility (H ⁄ D exchange) of a protein Moreover, the different kinetics of H ⁄ D exchange may assist in the assignment of absorption bands arising from different secondary structure classes [27,30–33] By contrast, IR experiments in D2O can also complicate the interpretation in the amide I¢ FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1717 Influence of inflammatory environment on HLA-B27 H Fabian et al 1718 Δ2nd derivative F E D C B 1652 1700 1600 Wavenumber (cm–1) 1516 1552 1595 1625 A 1694 1692 1668 Second derivative region, because spectral differences in the amide I¢ band contour due to H ⁄ D exchange and changes in secondary structure may overlap The only way to overcome this complication is to fully exchange the protein before monitoring conformational changes in D2O medium Complete H ⁄ D exchange is also achievable in the case of the HLA-B27:peptide complexes by keeping the sample solutions close to the denaturation temperature before cooling them to low temperature, but this is always accompanied by irreversible aggregation of the sample, thus rendering it impossible to obtain the IR spectrum of a completely exchanged native HLA-B27:peptide complex (data not shown, see also [27]) The interpretation of the IR spectra obtained in H2O medium is not complicated by the above-mentioned spectral effects due to H ⁄ D exchange We therefore also measured the IR spectra of the native HLA-B27:peptide complexes in H2O buffer, despite the fact that it is more difficult to obtain IR spectra in this solvent than in D2O-containing buffer because of the interfering intense water deformation band at around 1640 cm)1 [30] For a direct comparison with the measurements in D2O buffer described previously (Fig 2), the second derivatives of the absorbance spectra of the four complexes and their corresponding differences were calculated Interestingly, the IR spectra obtained in H2O buffer revealed subtype-specific spectral features (Fig 3) More importantly, many of these features in the amide I region resembled those described previously for the corresponding IR spectra in D2O buffer (compare Fig 2D,E with Fig 3A,C) Differences in peak position of the amide I bands at 1692 ⁄ 94 cm)1 assigned to the high-frequency b-sheet components, which also give rise to clear positive and negative features by subtracting the second derivative IR spectrum of B*2705:pVIPR-U5 from that of B*2709:pVIPR-U5 (Fig 3C), together with the spectral differences of the low-frequency b-sheet component at  1625 cm)1 were observed This corroborates the conclusions derived from analyses of the spectra in D2O buffer (Fig 2), that fine differences in the relative orientation of the strands in the b-sheet structures of the HC in the two HLA-B27 samples exist at low temperatures The components at  1650 cm)1 (Fig 3C) suggest a more intense and ⁄ or slightly shifted amide I band in the spectrum of B*2709 compared with that of B*2705 Again, the differences between the second derivative spectra of B*2705:pVIPR-U5 and B*2709:pVIPR-U5 turned out to be very similar to those found for the two HLA-B27:pVIPR complexes (compare Fig 3C with Fig 3D) This holds true for the different peak positions of the high-frequency b-sheet band around 1500 Fig HLA-B27 complexes measured in H2O buffer at pH 7.5 (Lower) IR spectra (second derivatives) of B*2709 ⁄ 13C-b2m and B*2705 ⁄ 13C-b2m (black and red traces in each panel, respectively), complexed with (A) pVIPR-U5 or (B) pVIPR at 15 °C (Middle) Differences between the second derivative IR spectra of the HLAB27:peptide complexes all measured at 15 °C (C) B*2709 ⁄ 13 C-b2m:pVIPR-U5 ) B*2705 ⁄ 13C-b2m:pVIPR-U5 and (D) B*2709 ⁄ 13 C-b2m:pVIPR ) B*2705 ⁄ 13C-b2m:pVIPR (Upper) IR-difference spectra of the second derivatives of experiments with two independent preparations of each HLA-B27:peptide complex at 15°C (E) complexed with pVIPR-U5 (black trace, B*2709; red trace, B*2705) and (F) complexed with pVIPR (black trace, B*2709; red trace, B*2705) The spectra of the samples were normalized by use of the tyrosine absorption band at 1516 cm)1 as an internal intensity standard 1690 cm)1 and the spectral differences between the features at  1650 and  1626 cm)1 as well Moreover, the subtype-dependent spectral differences were much more pronounced than the spectral differences between two independent preparations of the corresponding complexes (Fig 3E,F), as demonstrated previously for the corresponding IR spectra in D2O buffer (Fig 2F) [27] Altogether, the high degree of similarity between the corresponding IR spectra in D2O and in H2O buffer permits us to conclude that the spectroscopic FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS H Fabian et al Influence of inflammatory environment on HLA-B27 features in the amide I ⁄ I¢ regions associated with the polypeptide backbone both indicate subtle subtype-specific structural differences, rather than being the consequence of minor subtype-specific differences in H ⁄ D exchange of the amide protons in the corresponding HLA-B27:peptide complexes h after transfer into D2O buffer These subtype-specific structural differences might influence temporary local or global unfolding of the HC, and thus its flexibility pH-dependent thermal stabilities of peptide-complexed HLA-B27 subtypes C Intensity change (1592 cm–1) Having established that HLA-B27 subtype-specific, but peptide sequence-independent, conformational differences between the two HC exist in solution, we next investigated whether the presence of a hydrogen bond between the pU5 side chain and His116 of the HC might impact on the thermal denaturation behaviour of the HLA-B27:pVIPR-U5 complexes at physiological pH Following our previous approach [27], we employed the aromatic ring-stretching vibration of the tyrosine band at 1514 cm)1 to follow temperature-induced conformational changes in the HC The decrease in absorbance at 1592 cm)1 with increasing temperature was used to monitor denaturation of the secondary structure of b2m in the complexes The various frequency ⁄ temperature plots obtained by monitoring the tyrosine ring vibration (Fig 4A) revealed very similar thermostabilities of the HC in the two complexes at pH 7.5 (Table 1) The thermal denaturation temperatures estimated from the 13C-labelled b2m band of the two HLA-B27:pVIPR-U5 complexes were almost identical (Fig 4C), with a weak tendency to reach higher Tm values than those determined for the HC-specific tyrosine band (Table 1) Moreover, the transition temperature of b2m in the two complexes ( 64 °C) was very similar to that of free 13C-labelled b2m ( 65 °C) (Table 1) By contrast to this finding, distinct thermal denaturation temperatures were observed for the two HLAB27 subtypes complexed with the unmodified peptide pVIPR (Fig 4B,D) The B*2709 ⁄ 13C-b2m:pVIPR complex was less thermostable than the B*2705 ⁄ 13 C-b2m:pVIPR complex by 4–5 °C Moreover, a difference in peak position of the tyrosine band between the spectra of B*2709 ⁄ 13C-b2m:pVIPR and B*2705 ⁄ 13 C-b2m:pVIPR in the temperature range from 15 to D 10 20 30 40 50 60 70 80 90 A 10 20 30 40 50 60 70 80 90 B 1514.8 1514.6 1514.6 1514.4 1514.4 1514.2 1514.2 1514.0 Peak position (cm–1) 1514.8 1514.0 10 20 30 40 50 60 70 80 90 Temperature (°C) 10 20 30 40 50 60 70 80 90 Temperature (°C) Fig Thermostabilities of HLA-B27:peptide complexes at pH 7.5 All measurements were carried out in D2O buffer I and monitored by IR spectroscopy For each plot, the first data point (at 15 °C) was obtained h after transfer of the corresponding sample into D2O buffer The temperature dependence of the position of the HC-specific tyrosine band at 1514 cm)1 is shown for (A) B*2709 ⁄ 13C-b2m:pVIPR-U5 (d) and B*2705 ⁄ 13C-b2m:pVIPR-U5 (s), as well as for (B) B*2709 ⁄ 13C-b2m:pVIPR (d) and B*2705 ⁄ 13C-b2m:pVIPR (s) The other panels depict the temperature dependence of the peak intensity of the b2m-specific b-sheet band at 1592 cm)1 for (C) B*2709 ⁄ 13C-b2m:pVIPR-U5 (d) and B*2705 ⁄ 13C-b2m:pVIPR-U5 (s) as well as for (D) B*2709 ⁄ 13C-b2m:pVIPR (d) and B*2705 ⁄ 13C-b2m:pVIPR (s) FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1719 Influence of inflammatory environment on HLA-B27 H Fabian et al Table Determination of the transition temperatures of HLA-B27:peptide complexes The transition temperatures (Tm values in °C) were calculated either from the intensity ⁄ temperature plot of the b-sheet band of b2m at 1592 cm)1 or from the frequency ⁄ temperature changes of the tyrosine ring vibration of the HC at 1514 cm)1 of the IR spectra of B*2705 ⁄ 13C-b2m:pVIPR–U5, B*2709 ⁄ 13C-b2m:pVIPR-U5, B*2709 ⁄ 13C-b2m:pVIPR and B*2705 ⁄ 13C-b2m:pVIPR h after transfer into D2O buffer Measurements were performed at pH 7.5 or 5.6 (see Materials and Methods for experimental details) For comparison, the transition temperatures as estimated from IR experiments with free 13C-labelled b2m are also shown The Tm values are the average of experiments with two or three independent preparations of each sample, with standard deviations of 0.5–1 °C IR band B*2705:pVIPR-U5 B*2709:pVIPR-U5 B*2705:pVIPR B*2709:pVIPR Tyr band b2m-band Tyr band b2m-band 63.0 63.8 63.4 63.4 64.7 64.4 63.2 63.7 65.9 66.4 62.3 63.8 61.0 62.5 52.8 55.9 C Intensity change (1594 cm–1) 50 °C was observed (Fig 4B), indicating that the microenvironment of at least some Tyr residues must differ between the two subtypes [27] In addition, the gain in stability of B*2709:pVIPR-U5 compared with B*2709:pVIPR (Table 1) is likely to be a consequence of an additional peptide–HC interaction (a hydrogen bond connecting pU5O7 and His116NE2) that is present only in B*2709:pVIPR-U5 This suspected involvement of His116 in stabilizing the B*2709 subtype complexed with pVIPR-U5, but not pVIPR, prompted us to study the effect of lowering the pH such that it approached that in an inflamed tissue [12] Peak position (cm–1) 64.8 48.7 pH 7.5 7.5 5.6 5.6 To this end, we analysed the influence of a pH value of 5.6 on the thermal stability of all four HLA-B27:peptide complexes (Fig 5) The data reveal that both HLA-B27:pVIPR-U5 complexes (Fig 5A,C) exhibited comparable and high thermostabilities with Tm values of  63 °C at pH 5.6 (Table 1) By contrast, and as suspected, a strong impact on the thermostability upon lowering the pH to 5.6 was observed for B*2709:pVIPR ( 10 °C), but not for B*2705:pVIPR (Table 1) The lack of a comparable pH-induced decrease in thermal stability in case of B*2709:pVIPR-U5 allows to conclude that: (a) it is likely that the hydrogen bond between the D 10 20 30 40 50 60 70 80 90 A b2m 10 20 30 40 50 60 70 80 90 B 1514.8 1514.8 1514.6 1514.6 1514.4 1514.4 1514.2 1514.2 1514.0 1514.0 1513.8 1513.8 1513.6 10 20 30 40 50 60 70 80 90 Temperature (°C) 10 20 30 40 50 60 70 80 90 Temperature (°C) Fig Thermostabilities of the HLA-B27:peptide complexes at pH 5.6 All measurements were carried out in D2O buffer II and monitored by IR spectroscopy For each plot, the first data point (at 15 °C) was obtained h after transfer of the corresponding sample into D2O buffer The temperature dependence of the position of the HC-specific tyrosine band at 1514 cm)1 is shown for (A) B*2709 ⁄ 13C-b2m:pVIPR-U5 (d) and B*2705 ⁄ 13C-b2m:pVIPR-U5 (s), as well as for (B) B*2709 ⁄ 13C-b2m:pVIPR (d) and B*2705 ⁄ 13C-b2m:pVIPR (s) The other panels depict the temperature dependence of the peak intensity of the b2m-specific b-sheet band at 1594 cm)1 for (C) B*2709 ⁄ 13C-b2m:pVIPR-U5 (d) and B*2705 ⁄ 13C-b2m:pVIPR-U5 (s), as well as for (D) B*2709 ⁄ 13C-b2m:pVIPR (d) and B*2705 ⁄ 13C-b2m:pVIPR (s) 1720 FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS H Fabian et al pU5 side chain and His116 also exists in solution at physiological pH; and (b) the specific peptide–protein interaction involving His116 contributes to the conformational stability of the HLA-B27 complex Correlation of the results from IR spectroscopy with X-ray crystallographic data In an attempt to obtain a structure-based interpretation for the observed subtype-specific IR spectroscopic findings, we performed a detailed inspection of the X-ray structures of the two HLA-B27:pVIPR-U5 subtypes and analysed, in particular, the crystallographic temperature factors (B factors) of the different structural domains Because both HLA-B27:pVIPR-U5 subtypes crystallized isomorphously, we can exclude the possibility that differences between the two structures are due to different packing of the protein chains in crystallo A comparison of the X-ray structures revealed, however, that the structures of the HC and of b2m of the two subtypes appear nearly indistin˚ guishable (Ca root mean square deviation £ 0.6 A) [29] An earlier assessment of the B factors for the different structural units of B*2709:pVIPR and B*2705:pVIPR revealed that the peptide-binding groove exhibits the lowest flexibility, whereas major parts of the a3 domain and of b2m are more flexible [27] At the same time, this comparison failed to provide indications for differences between B*2709 and B*2705, which might serve to explain the observed dissimilarities in amide protection between the HC of the two subtypes In the case of complexes with the unmodified pVIPR peptide, this might have been due to the different resolutions at which the two structures ˚ ˚ were solved (B*2705 at 1.47 A, B*2709 at 2.2 A) [18] Such uncertainties not exist for the HLAB27:pVIPR-U5 subtypes, whose structures had been determined at high and comparable resolutions of ˚  1.8 A [29] An inspection of the binding grooves of B*2705:pVIPR-U5 and B*2709:pVIPR-U5, colourcoded according to the binding groove flexibility in the crystalline state at 100 K, revealed no clear indications for subtype-specific differences In summary, neither the comparison of the crystallographic temperature factors nor the detailed comparison of structural features provide hints which could help to understand the IR spectroscopic findings observed in solution at physiological temperatures Discussion This study addresses questions that are relevant for understanding how an inflammatory environment, Influence of inflammatory environment on HLA-B27 such as that observed in reactive arthritis or AS, might affect MHC molecules: (a) Is the HC flexibility of two minimally distinct HLA-B27 subtypes affected by citrullination of peptides? and (b) Can the stability of HLA-B27:peptide complexes be impaired by lowering the pH to levels which prevail in inflamed tissues? The isotope-edited IR spectroscopic results described here corroborate the previously observed differential flexibility of the two HLA-B27 subtypes To date, we had analysed only peptides (pVIPR, TIS, pLMP2) that are bound to B*2709 in the canonical binding mode, with the middle of the peptide bulging out of the binding groove [18,21,22,27,28] This, however, is not the case with pVIPR-U5, because the pArg5 fi pU5 exchange leads to a reorientation of this peptide in the binding groove of the B*2709 subtype, accompanied by the creation of a novel hydrogen-mediated contact between citrulline and His116 (Fig 1) [29] Therefore, the increased conformational flexibility of the B*2705 HC in comparison to that of B*2709 found also in case of pVIPR-U5 (see IR results, Fig 2) must be regarded as an intrinsic property of the HC of the two subtypes and not as a peptide sequence- or binding mode-related characteristic, particularly because this modified peptide is bound in a single, canonical conformation by B*2705 Ultimately, the polymorphic HC residue 116 must be responsible for the observed reorientation of pVIPR-U5 in comparison with the pVIPR peptide (Fig 6) We have already proposed a structure-based explanation to account for effects observed in conjunction with the Asp116His exchange, suggesting that a repositioning of water molecules is responsible for the altered flexibility of the two opposing helical segments of the binding groove [28] IR spectroscopy cannot be used to localize the regions where the two HC differ, but molecular dynamics simulations of complexes of HLA-B27 subtypes with pVIPR have suggested an increased flexibility of two opposing helical segments (residues 75–60 and 137–150) of the B*2705 binding groove in comparison with that of B*2709 [27] Corresponding MD simulations of the two HLA-B27 subtypes with the modified peptide pVIPR-U5 have not been carried out, but the high degree of similarity of the subtype-specific spectral differences for B*2705 ⁄ B*2709 either with pVIPR or pVIPR-U5 as observed in this study (Figs and 3) argues for a comparable nature of the underlying structural differences The observed subtype-specific differences in the IR b-sheet spectral features of the HC could not be explained on the basis of their X-ray structures, because all b strands of the B*2705 and B*2709 HC FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1721 Influence of inflammatory environment on HLA-B27 H Fabian et al Fig Conserved interactions between the displayed peptide and amino acid residues residing on the a1- or a2 helix All structures are superimposed and the view is towards the carboxylate of the C-terminal p9 residue Only the peptide segments from p5 to p9 are shown, and side chains are omitted with the exception of pArg5 (pVIPR) and pU5 (pVIPR-U5) HC residues Asp77 and Trp147, which are involved in conserved interactions with peptide residues, are shown as grey sticks Hydrogen-bonding interactions of the peptide residues p8 (main chain carbonyl) to Trp147 (indole NE atom) and of p9 (main chain amide) to Asp77 (carboxylate OD1 atom) are depicted with red dashed lines (A) B*2705:pVIPR in canonical conformation (green) and in noncanonical conformation (orange) Only the latter conformation allows the peptide to anchor to the HC by a salt bridge from pArg5 to Asp116 The conformation of pVIPR in B*2709 (brown) is indistinguishable from the canonical binding mode of this peptide in B*2705 (B) B*2705 in complex with pVIPR-U5 is shown in purple and B*2709 with pVIPR-U5 in yellow In the latter complex, a hydrogen bond is formed between His116 and pU5 The pU5 side chain points to different directions in the two subtypes Despite differences in peptide sequences and conformations, the formation of highly conserved hydrogen bonds from the a1- and a2 helices to the peptide main chain atoms is still permitted in all four structures complexed with pVIPR-U5 overlay perfectly Moreover, a comparative analysis of the B factors for the different structural domains provided no clear indications for differences between the HC of the two subtypes, suggesting that these conformational characteristics are only detectable in solution and thus inaccessible through X-ray data collection at cryogenic temperatures This might be because of the very limited information on protein dynamics that can be obtained at 100 K, or because the X-ray technique is not sensitive enough to resolve these differences As discussed previously [28], it seems plausible to assume that changes in the location of the bound water molecules near the helical regions may also impact on the conformation of the b sheet of the peptide-binding groove, which in turn may cause the spectral changes described Subtle spectral differences associated with b strands in proteins, which cannot easily be explained by X-ray crystallography, are not uncommon For example, we have shown before that a comparative IR spectroscopic analysis of the protein ribonuclease T1 and some of its variants can also reveal such alterations [34] The only difference observed by X-ray crystallography in these structures is a string of water molecules between the a-helix and the major b-sheet that are distinctly located in the wild-type protein and in the variants As pointed out previously [28], it is conceivable that peptides with a C-terminal basic residue 1722 might lead to an altered binding groove flexibility in the B*2705 subtype because of the formation of salt bridges to Asp116 within the molecule’s F pocket [17,35,36] This indicates that the conclusion reached with regard to the enhanced flexibility of the B*2705 HC may not be valid for those complexes that display a peptide with Arg or Lys at the C-terminus (see also [19,25,37] for further discussions) These considerations are irrelevant for the B*2709 subtype, however, because peptides with basic C-termini bind only very rarely to this subtype in vivo [4,16] How distinct dynamic characteristics of the two HLA-B27 subtypes impact on their function is currently unknown We have previously argued that the interaction of these molecules with receptors on effector cells might be altered in dependence on HC flexibility [28] However, in the absence of thorough analyses of dynamic properties concerning entire assemblies of MHC class I molecules and receptors on T cells or natural killer cells, it is currently not clear whether interactions of the binding partners are indeed influenced Analysis of a T-cell receptor footprint on an HLA-A2:peptide complex by NMR spectroscopy [38] is a first step towards understanding this intricate issue, although the flexibility of the MHC molecule was not investigated in this study In addition to the general subtype-specific conformational features discussed above, our experimental find- FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS H Fabian et al Influence of inflammatory environment on HLA-B27 Fig Structural features of the pVIPR and pVIPR-U5 peptides bound to B*2705 and B*2709 The view is identical to that in Fig In all panels, the subtype-specific residue 116 is indicated in green The conserved His9 and His114 residues (magenta sticks) are involved in peptide–HC interactions via water molecules that are represented by red spheres Hydrogen bonds and salt bridges are depicted by dashed red lines (A) pVIPR-U5 drawn in purple as presented by B*2705 (B) pVIPR-U5 drawn in yellow as presented by B*2709 (C) The binding of pVIPR by B*2705 occurs in a dual conformation with roughly equal occupancy Although one of the binding modes resembles that found in B*2709 (green, compare Fig 6A), the other (orange) is distinct from the first between pLys3 and pTrp7 It is characterized by the formation of a salt bridge between pArg5 and Asp116 and is thus similar to the conformation of pVIPR-U5 when bound to B*2709 (compare B) (D) pVIPR (brown) is bound by B*2709 in a single conformation, with the middle of the peptide bulging out of the binding groove Note that hydrogen bonds involving His9, His114 and His116 (only B*2709) are retained in a nearly identical manner by the two complexes of each HLA-B27 subtype ings provide also information on the peptide–protein interaction involving His116 that is found only in B*2709:pVIPR-U5, and on its contribution to the stability of this complex The HC of B*2705 and B*2709 contain 10 and 11 histidine residues, respectively When complexed with pVIPR and pVIPR-U5, two (His9 ⁄ His114 in B*2705) or three (His9 ⁄ His114 ⁄ His116 in B*2709) are involved in peptide–HC interactions via water molecules in the two subtypes, as well as in direct interactions with the polymorphic residue 116 (Fig 7) The adopted side-chain rotamer is identical for each of the two or three histidines, and the interaction between His9 and pArg2 via a conserved water molecule is preserved in all four complexes also Furthermore, His114 is always located at hydrogenbonding distance from the polymorphic residue Asp ⁄ His116, and is therefore important for the proper coordination of the side-chain function of this polymorphic residue Additional indirect contacts are formed from His114 to the differently bound peptides In the structures of B*2705:pVIPR-U5 (Fig 7A) and B*2709:pVIPR (Fig 7D), His114 is connected via a conserved water molecule to pLys3, reflecting almost identical peptide-binding modes, as observed in both structures By contrast, in the structures of B*2709:pVIPR-U5 and B*2705:pVIPR (Fig 7B,C), His114 is involved in two indirect hydrogen-bond interactions with two different side-chain functions of the bound peptide In addition, the side chains of pU5 and pTrp7 (B*2709:pVIPR-U5) are connected via the same water molecule to the imidazole function of His114 (Fig 7B) Irrespective of the occurrence of the pVIPR peptide in a dual conformation (B*2705), a water molecule is establishing an indirect contact with FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1723 Influence of inflammatory environment on HLA-B27 H Fabian et al pLys3 in the canonical conformation or with pArg5 in the noncanonical binding mode Therefore, histidine residues within the binding groove of HLA-B27 molecules serve important functions in maintaining a bound peptide in place As indicated previously, it was to be expected that the distinct water networks within the HLA-B27 binding groove might be influenced by lowering the pH to values that predominate in an inflammatory milieu [12] Furthermore, the unique direct contact between pU5 and His116 (B*2709:pVIPR-U5) and the lack of it (B*2709:pVIPR) must be regarded as particularly prominent candidates for being affected by pH-induced changes The drastic difference ( 10 °C) found in the thermal stability between complexes of B*2709 with these two peptides at pH values of 7.5 and 5.6 (Fig and Table 1) demonstrates that the pU5–His116 contact [29] (a) is most likely present also at physiological temperature in the noncrystalline state, and (b) stabilizes the B*2709:pVIPR-U5 complex As expected, the B*2705 subtype is not affected by the  100-fold elevated H+ concentration because of the replacement of His116 by Asp116 (Fig and Table 1) Experiments with further peptides and intermediate pH values will have to be carried out to assess the general relevance of these findings Nevertheless, it is already evident now that moderate changes in pH values within tissues have the potential to destabilize selected MHC:peptide complexes by influencing the protonation state of histidine residues, even when they are deeply embedded within the protein’s interior A decreased pH value (5 or lower) is also typical for endosomal compartments in which the loading of MHC class II antigens and CD1 molecules (b2m-complexed, lipid-binding MHC class I-like proteins) is accomplished [39,40] A histidine residue within the a1-domain of class II antigens (His33) has been suggested to be primarily responsible for facilitating the peptide exchange that is necessary for the loading of MHC class II molecules and the initiation of immune responses [41] To the best of our knowledge, the results presented here are the first to provide hints of the involvement of a naturally occurring MHC polymorphism that exerts a pH-dependent effect on the conformational stability of an MHC molecule, although the suggested protonation of His116 in B*2709:pVIPR complexes must be regarded as indirect and needs to be substantiated by further experiments Direct proof for the protonation of individual histidine residues might be obtained by neutron diffraction techniques, as revealed by the extremely complex picture of differential protonation for the 38 histidine residues of the haemoglobin tetramer [42] 1724 Materials and methods Sample preparation HPLC-purified, citrulline-modified peptide pVIPR-U5 was purchased from Alta Bioscience (Birmingham, UK) The nonapeptide pVIPR was synthesized and purified by M Beyermann (Leibniz-Institut fur Molekulare Pharmakologie, ă Berlin, Germany) The extracellular domains of B*2705 and B*2709 and 13C-labelled b2m were expressed separately in Escherichia coli as inclusion bodies [17,43] To obtain 13 C-labelled b2m, E coli cells were cultured in minimal medium containing a mixture of trace minerals, ammonium chloride and uniformly 13C-labelled glucose (Cambridge Isotope Labs, Andover, MA, USA), as described previously [27] For complex formation, the proteins were solubilized with urea, and refolded by dilution in the presence of the peptide [17,43] Following reconstitution, the entire mixture was concentrated using Amicon Ultra-15 devices of 10-kDa molecular weight cutoff (Millipore Corp., Billerica, MA, USA), and the complexes were isolated by size-exclusion chromatography in 10 mm sodium phosphate buffer, pH 7.5, 150 mm NaCl For the IR measurements, the sample solutions were exchanged at room temperature with the corresponding D2O buffer (buffer I: 10 mm sodium phosphate buffer, pH 7.5, 150 mm NaCl; buffer II: 100 mm MES pH 5.6) using Vivaspin 500 concentrators (Sartorius AG, Gottingen, Germany) with a membrane of 10- and ă 5-kDa molecular mass cut-off for the complexes and for free b2m, respectively This procedure took  40 for each sample The final sample concentrations were between 10 and 20 mgỈmL)1 before collection of IR data of the complexes and  mgỈmL)1 for b2m IR spectroscopy The protein solutions were always freshly prepared and placed into demountable calcium fluoride IR cells [30] with an optical pathlength of 50 lm for measurements in D2O buffer or lm for samples in H2O buffer IR spectra were recorded with IFS-28B and IFS-66 FTIR spectrometers (Bruker Optics, Ettlingen, Germany) equipped with deuterated triglycine sulfate detectors and continuously purged with dry air For each sample, 128 interferograms were co-added and Fourier-transformed to yield spectra with a nominal resolution of cm)1 The sample temperature was controlled by means of thermostated cell jackets Spectra at discrete temperatures were obtained by heating the protein solutions from 15 to 90 °C in steps of 2.5 °C In order to minimize problems due to baseline drifts or variations in the dry air purging of the spectrometer, the sample in the cell jacket was mounted on a motor-driven sample shuttle This allowed recording of the background immediately before recording of the sample spectrum without opening the sample chamber of the spectrometer Buffer spectra FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS H Fabian et al were recorded under identical conditions and subtracted from the spectra of the proteins in the relevant buffer and at the relevant temperature Spectral contributions from residual water vapour, if present, were eliminated using a set of water vapour spectra The final unsmoothed protein spectra were used for further analysis Band positions and band intensities were determined by standard functions of the Bruker OPUS software, implemented into home-built macros for data analysis Second derivatives were obtained using the Savitzky–Golay algorithm with 13-point smoothing Intensity ⁄ temperature and frequency ⁄ temperature plots of the IR data were created using origin software and analysed by non-linear fitting procedures [27] to estimate the thermal midpoint of a transition Acknowledgements We are grateful to Dr Michael Beyermann, LeibnizInstitut fur Molekulare Pharmakologie, Berlin, ă Germany, for synthesis and purification of the peptide pVIPR, and to Christina Schnick for excellent technical assistance We thank Dr Markus Wahl for continuous encouragement and support This work was supported by the Deutsche Forschungsgemeinschaft (grants Na226 ⁄ 12-3, UC8 ⁄ 1-2, and SFB 449 ⁄ B6), and a grant from the Robert Koch-Institut, Berlin (to Hans Huser) Andreas Ziegler also acknowledges support from the 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and presentation Nat Rev Immunol 8, 607–618 41 Rotzschke O, Lau JM, Hofstatter M, Falk K & ă ă Strominger JL (2002) A pH-sensitive histidine residue as control element for ligand release from HLA-DR molecules Proc Natl Acad Sci USA 99, 16946–16950 42 Kovalevsky AY, Chatake T, Shibayama N, Park SY, Ishikawa T, Mustyakimov M, Fisher Z, Langan P & Morimoto Y (2010) Direct determination of proton˚ ation states of histidine residues in a A neutron structure of deoxy-human normal adult haemoglobin and implications for the Bohr effect J Mol Biol 398, 276– 291 43 Garboczi DN, Hung DT & Wiley DC (1992) HLAA2–peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides Proc Natl Acad Sci USA 89, 3429–3433 FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS H Fabian et al Supporting information The following supplementary material is available: Fig S1 IR absorbance spectra of a B*2709 ⁄ 13Cb2m:pVIPR-U5 complex in H2O buffer, h after transfer into D2O buffer (partially H ⁄ D exchanged state), and in D2O buffer after cooling from 90 to 25 °C (fully deuterated state) This supplementary material can be found in the online version of this article Influence of inflammatory environment on HLA-B27 Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1727 ... drastically different conformations In addition, pVIPR is bound in a canonical single conformation by B*2709, but in an exceptional dual conformation by the AS- associated B*2705 subtype One of. .. protonation of ionizable groups in folded proteins may contribute to their conformational stability [14] Among the HLA-B27 subtypes, HLA-B*2705 (in short, B*2705) is strongly associated with AS, ... compilation ª 2011 FEBS 1719 Influence of inflammatory environment on HLA-B27 H Fabian et al Table Determination of the transition temperatures of HLA-B27: peptide complexes The transition temperatures