44eda31289c831aea06c34587cd79026 ijms-26-09026.pdf bc47997d32743de7bd07a3cf46a03ffd94eb0bf3 ijms-26-09026.pdf 40f921f077787cf3c364d606d4254ea6ea577d49f4b66590b332a3061a583b99 ijms-26-09026.pdf Title: Structural Study of a Peptide Epitope Bearing Multiple Post-Translational Modifications in Rheumatoid Arthritis Subject: Given the limited knowledge of the effect of post-translational modifications (PTMs) on protein structure, in this study we investigated whether introducing one-to-three RA-related PTMs into the -fibrin (617–631) peptide influences the conformation and structure of the peptide antigen that could be responsible for the autoantibody recognition. Ten peptides containing a different number of PTMs within their primary structure were synthesized and their recognition by sera from RA patients was analyzed. The conformation of the peptides was studied by circular dichroism (CD) and the structure of the most relevant antigenic peptides was determined by nuclear magnetic resonance (NMR) and enhanced-sampling molecular dynamics (MD). Although peptides containing citrulline (Cit) showed a higher degree of binding to AMPAs than peptides containing only homocitrulline and/or acetyl-lysine, the latter were able to bind to AMPAs in sera that showed a small response to peptides with Cit, with the response being different depending on the position of each PTM. CD and NMR analyses indicated a series of half-turn conformations in the Lys620-Arg630 region. MD simulations generated a set of conformations compatible with the NMR NOEs. The effect of the PTMs was observed in intra-molecular contacts, hydrogen bonds and van der Waals interactions, generating more collapsed conformations. Differences in autoantibody reactivity between peptides bearing different PTMs within their primary structures are noted. Peptides with PTMs adopt different conformations than unmodified peptides, probably due to the lower net charge of peptides with multiple PTMs, which may explain their recognition by autoantibodies. Keywords: RA; -fibrin (617–631) peptides; PTMs; AMPAs; CD; NMR; MD; conformation; structure Author: María José Gómara, Cristina García-Moreno, Oriol Bárcenas, Raúl Castellanos-Moreira, Juan Camilo Sarmiento, Ramon Crehuet, Yolanda Pérez, Raimon Sanmartí and Isabel Haro Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Wed Sep 17 07:48:08 2025 CEST ModDate: Wed Sep 17 07:51:36 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2331288 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CTPJRC+URWPalladioL-Ital Type 1 Custom yes yes yes 137 0 MIKRSI+VnURWPalladioL Type 1 Custom yes yes yes 138 0 VOPEOJ+URWPalladioL-Bold Type 1 Custom yes yes yes 139 0 FOBRAS+EURM10 Type 1 Builtin yes yes yes 140 0 YUIZQQ+URWPalladioL-Roma Type 1 Custom yes yes yes 141 0 KBKNOM+PalatinoLinotype TrueType WinAnsi yes yes no 195 0 KBKNOK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 196 0 KBKPEK+TimesNewRoman TrueType WinAnsi yes yes no 214 0 KBKNOM+PalatinoLinotype TrueType WinAnsi yes yes no 215 0 QIELJB+CMMI10 Type 1 Builtin yes yes yes 265 0 KBKNOO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 237 0 KBKNOM+PalatinoLinotype TrueType WinAnsi yes yes no 238 0 KBKNOO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 260 0 KBKNOM+PalatinoLinotype TrueType WinAnsi yes yes no 261 0 KBKNOM+PalatinoLinotype TrueType WinAnsi yes yes no 293 0 MPUDFL+PazoMath Type 1 Builtin yes yes yes 358 0 IHGURL+EURM7 Type 1 Builtin yes yes yes 359 0 OJYQXC+CMSY10 Type 1 Builtin yes yes yes 360 0 KBKNOM+PalatinoLinotype TrueType WinAnsi yes yes no 345 0 KBKNOM+PalatinoLinotype TrueType WinAnsi yes yes no 352 0 KBKNOM+PalatinoLinotype TrueType WinAnsi yes yes no 387 0 KBKNOM+PalatinoLinotype TrueType WinAnsi yes yes no 418 0 KBKNOM+PalatinoLinotype TrueType WinAnsi yes yes no 436 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-02-17 04:10:37 CET RepresentationInformation: ijms-26-09026.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-02-16 16:00:36 CET Size: 2331288 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 616 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Results and Discussion Children: Item: Title: Peptide Synthesis Item: Title: Recognition of the Different Epitopes by RA Patients’ Sera Item: Title: Reactivity to Peptides Containing a Single PTM Item: Title: Recognition of Peptides Containing Multiple PTMs Item: Title: Conformational and Structural Studies Children: Item: Title: Circular Dichroism Conformational Studies Item: Title: Nuclear Magnetic Resonance Structural Studies Item: Title: Molecular Dynamics Simulations Item: Title: Materials and Methods Children: Item: Title: Synthesis, Purification and Characterization of Peptides Item: Title: Home-Designed ELISA Assays Item: Title: Circular Dichroism Assays Item: Title: NMR Methodology Item: Title: Molecular Dynamics Item: Title: Conclusions Item: Title: References Info: Title: Structural Study of a Peptide Epitope Bearing Multiple Post-Translational Modifications in Rheumatoid Arthritis Author: María José Gómara, Cristina García-Moreno, Oriol Bárcenas, Raúl Castellanos-Moreira, Juan Camilo Sarmiento, Ramon Crehuet, Yolanda Pérez, Raimon Sanmartí and Isabel Haro Subject: Given the limited knowledge of the effect of post-translational modifications (PTMs) on protein structure, in this study we investigated whether introducing one-to-three RA-related PTMs into the -fibrin (617–631) peptide influences the conformation and structure of the peptide antigen that could be responsible for the autoantibody recognition. Ten peptides containing a different number of PTMs within their primary structure were synthesized and their recognition by sera from RA patients was analyzed. The conformation of the peptides was studied by circular dichroism (CD) and the structure of the most relevant antigenic peptides was determined by nuclear magnetic resonance (NMR) and enhanced-sampling molecular dynamics (MD). Although peptides containing citrulline (Cit) showed a higher degree of binding to AMPAs than peptides containing only homocitrulline and/or acetyl-lysine, the latter were able to bind to AMPAs in sera that showed a small response to peptides with Cit, with the response being different depending on the position of each PTM. CD and NMR analyses indicated a series of half-turn conformations in the Lys620-Arg630 region. MD simulations generated a set of conformations compatible with the NMR NOEs. The effect of the PTMs was observed in intra-molecular contacts, hydrogen bonds and van der Waals interactions, generating more collapsed conformations. Differences in autoantibody reactivity between peptides bearing different PTMs within their primary structures are noted. Peptides with PTMs adopt different conformations than unmodified peptides, probably due to the lower net charge of peptides with multiple PTMs, which may explain their recognition by autoantibodies. 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