Probing Functional Heteromeric Chemokine Protein–Protein Interactions through Conformation‐Assisted Oxime Ligation

Abstract Protein–protein interactions (PPIs) govern most processes in living cells. Current drug development strategies are aimed at disrupting or stabilizing PPIs, which require a thorough understanding of PPI mechanisms. Examples of such PPIs are heteromeric chemokine interactions that are potentially involved in pathological disorders such as cancer, atherosclerosis, and HIV. It remains unclear whether this functional modulation is mediated by heterodimer formation or by the additive effects of mixed chemokines on their respective receptors. To address this issue, we report the synthesis of a covalent RANTES‐PF4 heterodimer (termed OPRAH) by total chemical synthesis and oxime ligation, with an acceleration of the final ligation step driven by PPIs between RANTES and PF4. Compared to mixed separate chemokines, OPRAH exhibited increased biological activity, thus providing evidence that physical formation of the heterodimer indeed mediates enhanced function.

Abstract: Protein-protein interactions (PPIs) govern most processes in living cells.C urrent drug development strategies are aimed at disrupting or stabilizing PPIs,w hichr equire at horough understanding of PPI mechanisms.E xamples of such PPIs are heteromeric chemokine interactions that are potentially involved in pathological disorders such as cancer, atherosclerosis,a nd HIV.I tr emains unclear whether this functional modulation is mediated by heterodimer formation or by the additive effects of mixed chemokines on their respective receptors.T oa ddress this issue,w er eport the synthesis of ac ovalent RANTES-PF4 heterodimer (termed OPRAH) by total chemical synthesis and oxime ligation, with an acceleration of the final ligation step driven by PPIs between RANTES and PF4. Compared to mixed separate chemokines,O PRAH exhibited increased biological activity, thus providing evidence that physical formation of the heterodimer indeed mediates enhanced function.
Chemokines are small chemotactic cytokines that mediate leukocyte trafficking,which makes them interesting pharmaceutical targets to antagonize inflammatory diseases such as rheumatoid arthritis,C rohnsd isease,a nd atherosclerosis. [1] By binding to their cognate Gprotein-coupled receptors,they can rapidly trigger leukocyte chemotaxis and adhesion, and thus regulate av ariety of cellular processes for example, angiogenesis,e mbryonic development, and cell homeostasis. [2] Thestructure of chemokine monomers is characterized by ad isordered N-terminus and three-stranded antiparallel bsheets onto which aC-terminal a-helix is folded. Based on the spacing between conserved N-terminal cysteiner esidues, chemokines are divided into four groups,w ith the major ones being the CC-and CXC-types.A lthough small (< 10 kDa), most chemokines have the propensity to form homodimers or higher-order multimeric structures (> 200 kDa). [3] While the monomeric structures of all chemokines are highly conserved, there are notable differences between their (oligomeric) quaternary structures.F or example,C C-type chemokine dimers,f ormed by interactions between their N-termini, are elongated and dumbbell shaped, whereas CXC-type chemokine dimers are formed by interactions between their central b-sheets,w hich makes them more globular in structure.
Thec hemokines RANTES (CCL5) and platelet factor 4 (PF4, CXCL4) are stored in the a-granules of platelets.While RANTES is ap otent attractant of mononuclear cells,P F4 lacks classical chemo-attractant functions. [4] However,t he presence of PF4 can synergistically enhance RANTESmediated cell-recruitment mediated by heterophilic interactions between chemokines. [4] In fact, pharmacologic inhibition of RANTES and PF4 heterodimer formation inhibits monocyte recruitment and attenuates progression of atherosclerosis,lung injury,and aneurysm formation in mice. [5] Heterophilic chemokine interactions have also been reported for CXCL4 and CXCL8, [6] CCL19 and CCL22, [7] CCL2 and CCL8, [8] as well as other chemokines. [9] These studies support the proposal that heterophilic interactions between different chemokines are relevant for many physiological and pathological processes.
Although current evidence suggests that physical formation of chemokine heterodimers mediates their observed functional modulation, the possibility remains that this could simply be due to combined receptor stimulation by the respective chemokines.T herefore,t he aim of the present study is to generate an obligate RANTES-PF4 heterodimer by total chemical synthesis to address this crucial mechanistic question.
Based on the observation that RANTES and PF4 interact through their N-termini as aC C-type dimer, [5a] we used this model as astarting point to assess optimal coupling strategies for the protein pair ( Figure 1). Because oxime ligation can be performed with folded proteins in aqueous buffer at neutral pH, we chose it to covalently link the two proteins.O xime ligation requires the incorporation of aketone in one protein and an aminooxy moiety in the other. Owing to limitations in the ketone functionality of the widely-used levulinic acid, we used 5-ketohexanoic acid because of its high reactivity and conformational flexibility. [10] Theproposed heterodimer consists of aRANTES monomer (68 aa) that is covalently linked to aP F4 monomer (76 aa) (Figure 2). Thec hemokines were each assembled by two subsequent native chemical ligations (NCL), [11] followed by oxidative folding,a nd joined by oxime ligation. Positions suitable for covalent linkage of the two proteins were based on the proposed heterodimer structure observed by NMR spectroscopy.
[5a] Furthermore,t he N-terminus of RANTES was required to remain unrestricted to allow interactions with its receptor (CCR5). [12] Additionally,a mino acids crucial for RANTES-PF4 interaction were left untouched, leaving Thr7 in RANTES and Leu8 in PF4 as as ite for crosslinking ( Figure 1). Therefore,T hr7 and Leu8 were replaced by orthogonal protected lysines to provide ah andle for intro-  . Both C-terminalp eptides (3 and 11)w ere synthesized using standard Boc solid-phase peptide synthesis protocols. [13] Sequential NCL required N-terminal encrypted cysteines (thiaprolines) to be used in the middle fragments (2 and 10). [14] Both Nterminal fragments (1 and 9)c ontain athioester for NCL and require an orthogonally protected (Ne-Alloc) lysine for incorporation of the ketone or aminooxy functional group. Deprotection of the Alloc group was performed on resin using Pd(PPh 3 ) 4 ,and the ketone (5-ketohexanoic acid) and aminooxy (Boc-(aminooxy)acetic acid) moieties were introducedt oobtain 5 and 13,respectively.After synthesis and purification, all peptide fragments were ligated sequentially using 1-2 %t hiophenol and benzylmercaptan as catalysts.

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Communications ducing the ketone or aminooxy moiety.A ll reactions were monitored by HPLC-MS.A fter oxidative folding of the chemokines to obtain modified RANTES (16)a nd PF4 (8), an oxime linkage between the two proteins was obtained by reacting equimolar quantities of the two modified chemokines (200 mm)i na queous buffer at pH 4.5. This led to the desired obligate PF4-RANTES heterodimer (OPRAH, 17) with a6 0% yield after 12 h ( Figure 2; Supporting Information, Figures S1 and S2). Af unctional negative control (nOPRAH) was established by linking Ser1 of RANTES to Glu1 of PF4 through an identical oxime linker by which the N-terminal interaction of RANTES with its receptor CCR5 was blocked.
In an effort to improve the reaction rate,weobserved only minor enhancement by addition of aniline as acatalyst. [15] To rule out that homomeric interactions between folded chemokines would impair heteromeric oxime formation, we performed oxime ligation in the presence of ad enaturing agent (6 m Gn·HCl). Surprisingly,i nstead of accelerating heteromeric oxime formation, the presence of Gn·HCl slowed down oxime ligation 5-fold, whereas it only had am inor effect on the reaction rate in am odel system ( Figure 3; Supporting Information, Figure S3). This observation indicated that the rate of formation of the oxime bond between RANTES and PF4 was accelerated by heterophilic chemokine interactions, which bring the two reactive species into close proximity ( Figure 3). In retrospect, the proposed conformation-assisted ligation was supported by the lower-than-expected catalytic effect of aniline.E ventually,u sing slow freezing catalysis, [16] we were able to achieve complete oxime reaction within 2h.
To assess the folded states of modified PF4 and RANTES, as well as OPRAH, we used NMR spectroscopy.N MR spectra show that modified PF4 and RANTES are well folded. However,modified PF4 could still form tetramers like wild type PF4, whereas modified RANTES with its Nterminally positioned lysyl-linker showed markedly attenuated dimer formation. Compared with the NMR spectra of the non-ligated proteins,that of OPRAH appears to show two correctly folded domains of the respective chemokines ( Figure 4, Supporting Information, Figure S4). Ther esolved Tr p57 He resonance in the spectrum of 16 is agood indicator of the folding preference of the RANTES subunit in OPRAH. In non-ligated RANTES,T rp57 He resonances are observed for monomer (m) and homodimer (d) states (inset, Figure 4). Upon covalent dimerization of 16 and 8 to form OPRAH (17), the RANTES homodimer ds tate peak decreases in intensity,w hereas the mp eak increases and has as light chemical shift, which is consistent with ac hange in chemical environment likely due to formation of the linked heterodimer.T he remaining RANTES dpeak intensity in 17 may arise from some homodimerization of two RANTES domains in OPRAH.
We also followed real-time oxime formation between 8 and 16 by NMR spectroscopy (Supporting Information, Figures S4-6). These NMR spectra show overall resonance broadening,w hich we attribute to transient formation of the OPRAH heterodimer.T his is supported by our observations with the He resonances of Tr p57, which is an indirect structural probe owing to its distance from the modified Nterminus of 16.Overall, we conclude that the general folds of the two protein domains,PF4 and RANTES,are maintained in OPRAH, and resonance broadening of the residues at the  Overlay of experimental 1 HNMR spectrum of OPRAH (black) and the sum of the spectra of free RANTES (16)and PF4 (8)(broken line) The agreement between experimental spectrum 17 and the summed spectra 8 + 16 shows that, globally,O PRAH consists of two natively folded PF4 and RANTES protein domains. Local differencesa re, however,apparent and these regions correspond to sharp amide peaks in the N-terminus region (amino acids 2-7) of free PF4 that seem to broaden out significantly in OPRAH. Inset:T rp57 He signal is indicated for free RANTES (16), the 1:1complex, and OPRAH (17).
www.angewandte.org oxime ligation site suggests dynamic interactions between the two covalently-linked domains.
We next investigated the biological activity of OPRAH in amonocyte arrest assay.Amonolayer of endothelial cells was pre-treated with chemokines or heterodimers,a fter which monocytes were passed over the surface. [4] Thep resence of chemokines triggered monocyte arrest, and the number of arrested monocytes was recorded as measure of activity ( Figure 5A). Thef ollowing three conditions were tested: OPRAH, the negative control nOPRAH (in which the Ntermini were blocked by oxime linkage at positions 1i n RANTES and PF4), and an on-covalent dimer-mix of RANTES and PF4. OPRAH (17)r ecruited twice as many monocytes as the non-covalent mixed PF4 and RANTES chemokines ( Figure 5B). Furthermore,t he negative control nOPRAH showed no activity,proving the importance of the free N-terminus of OPRAH for receptor-mediated monocyte recruitment. [12] Peptide CKEY,which was able to disrupt noncovalent PF4-RANTES heterodimers and led to adecrease in atherosclerosis in am ouse model, [5a] was able to reduce monocyte adhesion with non-covalent PF4-RANTES heterodimers but not with OPRAH ( Figure 5C).
In summary,w eh ave provided strong evidence that synergism in monocyte recruitment by the PF4-RANTES complex proceeds in a1 :1 heterodimeric fashion. Evidence for this concept through the chemical synthesis of acovalent PF4-RANTES heterodimer (OPRAH) additionally offers synthetic access to multiple chemokine heteromers that can unambiguously demonstrate functional enhancement through heteromerization within this novel area of chemokine biology.