Solution structure of CXCL13 and heparan sulfate binding show that GAG binding site and cellular signalling rely on distinct domains

Monneau, Yoan R.; Luo, Lingjie; Sankaranarayanan, Nehru Viji; Nagarajan, Balaji; Vivès, Romain R.; Baleux, Françoise; Desai, Umesh R.; Arenzana-Seidedos, Fernando; Lortat‐Jacob, Hugues

doi:10.1098/rsob.170133

Cited by 27 publications

(34 citation statements)

References 41 publications

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“…the residues after the C-terminal -helix) of CXCL13 is incredibly dynamic, with multiple trajectories away from the core domain. Comparisons of two other published structures of human CXCL13 bound to single-chain variable fragment (scFv) molecules (Tu et al, 2016) as well as two solution structures of murine CXCL13 (Monneau et al, 2017) confirmed our finding of the rigidity of the core domain as well as the mobilities of the N-and C-termini. Collectively, our results provide the scientific community with a case study as to how some chemokine receptors can tolerate minor length and side-chain variation in the N-termini of their ligands and retain activity, as well as providing two unique structures of human CXCL13.…”

Section: Introductionsupporting

confidence: 84%

“…other time: in the solution structure of murine CXCL13 (Monneau et al, 2017). Further inspection of Met CXCL13 indicated that there is one monomer per asymmetric unit that forms a crystallographic dimer with a symmetry mate.…”

Section: Crystal Structure Of Met Cxcl13mentioning

confidence: 99%

“…In addition to the structures of human CXCL13 in complex with scFvs described above, there also exist two structures of murine CXCL13 (mCXCL13) that have been solved by NMR (PDB entries 5l7m and 5izb; Monneau et al, 2017). To compare the differences between these structures and our own, we first performed an alignment of the primary sequences of both the human and murine variants using Clustal Omega (Madeira et al, 2019).…”

Section: Comparisons With Murine Cxcl13 Nmr Structuresmentioning

confidence: 99%

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The N-terminal length and side-chain composition of CXCL13 affect crystallization, structure and functional activity

Rosenberg

Herrington²,

Rajasekaran

et al. 2020

Acta Cryst Sect D Struct Biol

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CXCL13 is the cognate chemokine agonist of CXCR5, a class A G-protein-coupled receptor (GPCR) that is essential for proper humoral immune responses. Using a `methionine scanning' mutagenesis method on the N-terminus of CXCL13, which is the chemokine signaling region, it was shown that minor length alterations and side-chain substitutions still result in CXCR5 activation. This observation indicates that the orthosteric pocket of CXCR5 can tolerate these changes without severely affecting the activity. The introduction of bulk on the ligand was well tolerated by the receptor, whereas a loss of contacts was less tolerated. Furthermore, two crystal structures of CXCL13 mutants were solved, both of which represent the first uncomplexed structures of the human protein. These structures were stabilized by unique interactions formed by the N-termini of the ligands, indicating that CXCL13 exhibits substantial N-terminal flexibility while the chemokine core domain remains largely unchanged. Additionally, it was observed that CXCL13 harbors a large degree of flexibility in the C-terminal extension of the ligand. Comparisons with other published structures of human and murine CXCL13 validate the relative rigidity of the core domain as well as the N- and C-terminal mobilities. Collectively, these mutants and their structures provide the field with additional insights into how CXCL13 interacts with CXCR5.

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Section: Introductionsupporting

confidence: 84%

Section: Crystal Structure Of Met Cxcl13mentioning

confidence: 99%

Section: Comparisons With Murine Cxcl13 Nmr Structuresmentioning

confidence: 99%

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The N-terminal length and side-chain composition of CXCL13 affect crystallization, structure and functional activity

Rosenberg

Herrington²,

Rajasekaran

et al. 2020

Acta Cryst Sect D Struct Biol

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“…On the other hand, the role of CXCL13 as tissue-specific chemoattractant confined to lymph nodes and TLS, implicates the necessity for mechanisms restricting its diffusion and spatial distribution. The mechanisms for its tissue-specific local containment has recently been shown to rely on special molecular binding sites on CXCL13 for heparan sulfate side chains of proteoglycans on cells and within extracellular matrices [36]. These interactions are of utmost importance for chemokine gradients and haptotactic guidance of lymphocytes within tissues [37].…”

Section: Discussionmentioning

confidence: 99%

Chemokine CXCL13 in serum, CSF and blood–CSF barrier function: evidence of compartment restriction

Pilz

Sakic

Wipfler

et al. 2020

Fluids Barriers CNS

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Background and purpose: Elevation of the chemokine CXCL13 in CSF frequently occurs during active and acute CNS inflammatory processes and presumably is associated with B cell-related immune activation. Elevation levels, however, vary a lot and "leaking" of CXCL13 from blood across dysfunctional brain barriers is a possible source. The aim was to clarify the relation between CXCL13 concentrations in CSF, CXCL13 concentrations in serum and blood-CSF barrier (BCSFB) function for a correct interpretation of the intrathecal origin of CXCL13. Methods: We retrospectively analyzed CXCL13 of banked CSF/serum samples (n = 69) selected from patient records and categorized the CSF CXCL13 elevations as CXCL13 negative (< 30 pg/ml), low (30-100 pg/ml), medium (101-250 pg/ml), or high (> 250 pg/ml). CXCL13 concentrations in CSF and serum and the corresponding CSF/serum CXCL13 quotients (Qcxcl13) were compared to CSF/serum albumin quotients (QAlb) as a measure for BCSFB function. The CXCL13 negative category included two subgroups with normal and dysfunctional BCSFB. Results: Serum CXCL13 concentrations were similar across categories with median levels around 100 pg/ml but differed between individuals (29 to > 505 pg/ml). Despite clear evidence in serum, CXCL13 was detectable only at trace amounts (medians 3.5 and 7.5 pg/ml) in CSF of the two CXCL13 negative subgroups irrespective of a normal or pathological QAlb. Moreover, we found no association between CSF and serum CXCL13 levels or between QAlb and CSF CXCL13 levels in any of the CSF CXCL13-delineated categories. CXCL13 apparently does not "leak" from blood into CSF. This implies an intrathecal origin also for low CSF CXCL13 levels and a caveat for analyzing the Qcxcl13, because higher serum than CSF concentrations arithmetically depress the Qcxcl13 resulting in misleadingly low CSF/serum quotients. Conclusion: We demonstrated that CXCL13 does not cross from blood into CSF, not even during severe BCSFB dysfunction. CSF CXCL13 elevations therefore most likely always are CNS-derived, which highlights their relevance as indicator of inflammatory CNS processes. We recommend data should not be corrected for BCSFB permeability (QAlb) and not to calculate CSF/serum quotients for CXCL13 as these may introduce error.

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“…Our current studies and previous studies of hNACs show that residues from the N-loop and 40s loop also determine binding, that their contributions can be quite significant, and that the binding geometries can be quite diverse (24, 26 -29). Recent NMR studies of GAG binding of chemokines CXCL10, CXCL12, and CXCL13 also show diverse binding interfaces and geometries (41)(42)(43)(44). Considering all chemokines bind GAGs, it is very likely that each chemokine shows distinct GAG interactions.…”

Section: Discussionmentioning

confidence: 99%

Structural basis, stoichiometry, and thermodynamics of binding of the chemokines KC and MIP2 to the glycosaminoglycan heparin

Sepuru

Nagarajan

Desai

et al. 2018

Journal of Biological Chemistry

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Keratinocyte-derived chemokine (KC or mCXCL1) and macrophage inflammatory protein 2 (MIP2 or mCXCL2) play nonredundant roles in trafficking blood neutrophils to sites of infection and injury. The functional responses of KC and MIP2 are intimately coupled to their interactions with glycosaminoglycans (GAGs). GAG interactions orchestrate chemokine concentration gradients and modulate receptor activity, which together regulate neutrophil trafficking. Here, using NMR, molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC), we characterized the molecular basis of KC and MIP2 binding to the GAG heparin. Both chemokines reversibly exist as monomers and dimers, and the NMR analysis indicates that the dimer binds heparin with higher affinity. The ITC experiments indicate a stoichiometry of two GAGs per KC or MIP2 dimer and that the enthalpic and entropic contributions vary significantly between the two chemokine-heparin complexes. NMR-based structural models of heparin-KC and heparin-MIP2 complexes reveal that different combinations of residues from the N-loop, 40s turn, ␤ 3-strand, and C-terminal helix form a binding surface within a monomer and that both conserved residues and residues unique to a particular chemokine mediate the binding interactions. MD simulations indicate significant residue-specific differences in their contribution to binding and affinity for a given chemokine and between chemokines. On the basis of our observations that KC and MIP2 bind to GAG via distinct molecular interactions, we propose that the differences in these GAG interactions lead to differences in neutrophil recruitment and play nonoverlapping roles in resolution of inflammation. Chemokines, a large family of small molecular weight proteins, play crucial roles in the pathophysiology of infectious and inflammatory diseases (1-3). Common to these diverse func-This work was supported by National Institutes of Health Grants P01 HL107152, R21AI124681, and S10OD023576. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1-S3 and Movies S1 and S2.

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Solution structure of CXCL13 and heparan sulfate binding show that GAG binding site and cellular signalling rely on distinct domains

Cited by 27 publications

References 41 publications

The N-terminal length and side-chain composition of CXCL13 affect crystallization, structure and functional activity

The N-terminal length and side-chain composition of CXCL13 affect crystallization, structure and functional activity

Chemokine CXCL13 in serum, CSF and blood–CSF barrier function: evidence of compartment restriction

Structural basis, stoichiometry, and thermodynamics of binding of the chemokines KC and MIP2 to the glycosaminoglycan heparin

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