{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T10:49:00Z","timestamp":1785408540189,"version":"3.56.0"},"reference-count":39,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T00:00:00Z","timestamp":1778630400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003130","name":"Research Foundation Flanders","doi-asserted-by":"publisher","award":["G053822N"],"award-info":[{"award-number":["G053822N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003130","name":"Research Foundation Flanders","doi-asserted-by":"publisher","award":["I007220N"],"award-info":[{"award-number":["I007220N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003130","name":"Research Foundation Flanders","doi-asserted-by":"publisher","award":["1SC9822N"],"award-info":[{"award-number":["1SC9822N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003130","name":"Research Foundation Flanders","doi-asserted-by":"publisher","award":["1SC9824N"],"award-info":[{"award-number":["1SC9824N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003130","name":"Research Foundation Flanders","doi-asserted-by":"publisher","award":["1S11223N"],"award-info":[{"award-number":["1S11223N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003130","name":"Research Foundation Flanders","doi-asserted-by":"publisher","award":["1SF0324N"],"award-info":[{"award-number":["1SF0324N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]},{"award":["G053822N"],"award-info":[{"award-number":["G053822N"]}],"id":[{"id":"https:\/\/ror.org\/03qtxy027","id-type":"ROR","asserted-by":"publisher"}]},{"award":["I007220N"],"award-info":[{"award-number":["I007220N"]}],"id":[{"id":"https:\/\/ror.org\/03qtxy027","id-type":"ROR","asserted-by":"publisher"}]},{"award":["1SC9822N"],"award-info":[{"award-number":["1SC9822N"]}],"id":[{"id":"https:\/\/ror.org\/03qtxy027","id-type":"ROR","asserted-by":"publisher"}]},{"award":["1SC9824N"],"award-info":[{"award-number":["1SC9824N"]}],"id":[{"id":"https:\/\/ror.org\/03qtxy027","id-type":"ROR","asserted-by":"publisher"}]},{"award":["1S11223N"],"award-info":[{"award-number":["1S11223N"]}],"id":[{"id":"https:\/\/ror.org\/03qtxy027","id-type":"ROR","asserted-by":"publisher"}]},{"award":["1SF0324N"],"award-info":[{"award-number":["1SF0324N"]}],"id":[{"id":"https:\/\/ror.org\/03qtxy027","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004040","name":"KU Leuven","doi-asserted-by":"publisher","award":["C32\/18\/038"],"award-info":[{"award-number":["C32\/18\/038"]}],"id":[{"id":"10.13039\/501100004040","id-type":"DOI","asserted-by":"publisher"}]},{"award":["C32\/18\/038"],"award-info":[{"award-number":["C32\/18\/038"]}],"id":[{"id":"https:\/\/ror.org\/05f950310","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Background and objectives: The PWWP domain of lens epithelium-derived growth factor p75 (LEDGF\/p75) mediates chromatin engagement through recognition of histone H3 lysine 36 di- and trimethylation (H3K36me2\/3) and nucleosomal DNA. LEDGF\/p75 plays a role in multiple human diseases. In particular, its interaction with HIV-1 integrase enables viral genome integration. However, the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets. Here, we report the generation of high-affinity nanobodies (Nbs) to investigate the structure and function of this domain. Methods: Camelids were immunized with recombinant LEDGF PWWP domain, and immune phage display libraries were screened for affinity. Selected Nbs were recombinantly expressed in E. coli and purified. Their interaction with the PWWP domain of LEDGF and its close homolog HRP-2 was characterized using size-exclusion chromatography and surface plasmon resonance. Structural characterization of the Nbs was performed using X-ray crystallography. Functional effects on chromatin engagement were evaluated using an AlphaScreen assay. Results: Nine sequence-distinct Nbs were identified, seven of which were confirmed to bind the LEDGF PWWP domain with nanomolar affinities. Five Nbs also bound the HRP-2 domain, consistent with conserved functional surfaces, while two showed reduced affinity. The crystal structures of two Nbs (NbC03 and NbH10) confirmed there were canonical immunoglobulin folds, while the latter additionally revealed a domain-swapped dimer. Moreover, NbH10 dose-dependently inhibited the interaction between full-length LEDGF\/p75 and H3K36me3-modified nucleosomes in vitro. Conclusions: This work establishes a validated panel of Nbs targeting the LEDGF PWWP domain and identifies one Nb capable of functionally disrupting the LEDGF\u2013chromatin interaction. These Nbs serve as valuable tools for functional studies and structure-based drug design.<\/jats:p>","DOI":"10.3390\/biom16050716","type":"journal-article","created":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T16:46:18Z","timestamp":1779381978000},"page":"716","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["High-Affinity Nanobody Against the LEDGF PWWP Domain Inhibits Chromatin Binding In Vitro"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1452-9584","authenticated-orcid":false,"given":"Thibault","family":"Vantieghem","sequence":"first","affiliation":[{"name":"Biocrystallography, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sofie","family":"Jansen","sequence":"additional","affiliation":[{"name":"Biocrystallography, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thatcher Zinabu","family":"Akele","sequence":"additional","affiliation":[{"name":"Advanced Disease Modelling, Targeted Drug Discovery and Gene Therapy (ADVANTAGE), KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0460-1976","authenticated-orcid":false,"given":"Pieterjan","family":"Van Maele","sequence":"additional","affiliation":[{"name":"Laboratory for Therapeutic and Diagnostic Antibodies, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2831-5042","authenticated-orcid":false,"given":"Sam","family":"Noppen","sequence":"additional","affiliation":[{"name":"Molecular, Structural and Translational Virology, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3256-5850","authenticated-orcid":false,"given":"Dominique","family":"Schols","sequence":"additional","affiliation":[{"name":"Molecular, Structural and Translational Virology, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3138-281X","authenticated-orcid":false,"given":"Maarten","family":"Dewilde","sequence":"additional","affiliation":[{"name":"Laboratory for Therapeutic and Diagnostic Antibodies, KU Leuven, 3000 Leuven, Belgium"},{"name":"PharmAbs-The KU Leuven Antibody Center, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3982-1565","authenticated-orcid":false,"given":"Zeger","family":"Debyser","sequence":"additional","affiliation":[{"name":"Advanced Disease Modelling, Targeted Drug Discovery and Gene Therapy (ADVANTAGE), KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7189-4652","authenticated-orcid":false,"given":"Sergei V.","family":"Strelkov","sequence":"additional","affiliation":[{"name":"Biocrystallography, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"17308","DOI":"10.1073\/pnas.0506924102","article-title":"Structural Basis for the Recognition between HIV-1 Integrase and Transcriptional Coactivator P75","volume":"102","author":"Cherepanov","year":"2005","journal-title":"Proc. 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