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Distinct differences in structural states of conserved histidines in two related proteins: NMR studies of the chemokines CXCL1 and CXCL8 in the free form and macromolecular complexes Krishna Mohan Sepuru, and Krishna Rajarathnam Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00756 • Publication Date (Web): 19 Sep 2018 Downloaded from http://pubs.acs.org on September 20, 2018
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Biochemistry
Distinct differences in structural states of conserved histidines in two related proteins: NMR studies of the chemokines CXCL1 and CXCL8 in the free form and macromolecular complexes
Krishna Mohan Sepuru #, §, * and Krishna Rajarathnam#, §, * #Department
of Biochemistry and Molecular Biology, §Sealy Center for Structural Biology and
Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA. Running Title: Histidine side chain interactions
*Corresponding authors:
[email protected];
[email protected] 1 ACS Paragon Plus Environment
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Abstract Hydrogen-bonding and ionic interactions play fundamental roles in macromolecular recognition and function. In contrast to lysines and arginines, how histidines mediate these interactions is less well understood due to the unique properties of its side chain imidazole that include an aromatic ring with two titratable nitrogens, a pKa that can vary significantly, and the ability to exist in three distinct forms: protonated imidazolium and two tautomeric neutral (Nδ1 and Nε2) states. Here, we characterized the structural features of histidines in the chemokines CXCL8 and CXCL1 in the free, GAG heparinbound, and CXCR2 receptor N-terminal domain-bound states using solution NMR spectroscopy. CXCL8 and CXCL1 share two conserved histidines, one in the N-loop and the other in the 30s loop. In CXCL8, both histidines exist in the Nε2 tautomeric state in the free, GAG-bound, and receptor-bound forms. On the other hand, in unliganded CXCL1, each of the two histidines exists in two states, as the neutral Nε2 tautomer and charged imidazolium. Further, both histidines exclusively exist as the imidazolium in the GAG-bound and as the Nε2 tautomer in the receptor-bound forms. The N-loop histidine alone in both chemokines is involved in direct GAG and receptor interactions indicating the role of the 30s loop varies between the chemokines. Our observation that the structural features of conserved histidines and their functional role in two related proteins can be quite different is novel. We further propose that directly probing the imidazole structural features is essential to fully appreciate the molecular basis of histidine function.
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Introduction H-bonding and ionic interactions mediate macromolecular interactions and are fundamental to all physiological phenomena1-3. The basic amino acids lysine, arginine, and histidine are major drivers of H-bonding and electrostatic interactions. The side chain structures and properties such as pKa of these amino acids are quite different, indicating that how the side chain properties dictate different aspects of the binding, such as affinity and specificity, will be different. Whereas the lysine amine and arginine guanidinium groups are at the end of a long aliphatic chain and their pKa is >10, the imidazole side chain is cyclic with a pKa that can vary significantly. Whereas the pKa of a histidine in model peptides is ~6, pKa in proteins can vary between 10, indicating that the pKa is highly dependent on the local structure4-10. Further, the imidazole ring can exist in three forms due to two titratable nitrogens: protonated imidazolium and two tautomeric neutral (Nδ1 and Nε2) states11-14 (Figure 1). Accordingly, the role of histidines and the molecular basis of how histidine imidazole interacts with its cognate partner within the free protein and in complex formation can vary. g d1
e1
g d2
+
g
e1
e2
d1
d2
d1 e2
e1 e2 Nd1 tautomer
Ne2 tautomer
imidazolium
d2
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170
Ne2-He1 Ne2-He1
Ne2-Hd2
Nd1-He1
Nd1-Hd2
Ne2-Hd2
Nd1-He1
(ppm)
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15N
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
230
250
Ne2-He1
Nd1-He1
9.0
8.0
7.0
9.0 1H
8.0
7.0
9.0
Ne2-Hd2
8.0
7.0
(ppm)
Figure 1. A schematic of the neutral Nε2, neutral Nδ1, and charged imidazolium states of the histidine side chain and the expected 1H-15N HMQC spectrum of each species.
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Humans express ~ 50 chemokines, which play a crucial role in directing immune and non-immune cells in health and disease15. It is well established that chemokine function is intimately coupled to binding to two classes of macromolecules: glycosaminoglycans (GAGs) that are sulfated linear polysaccharides and receptors that belong to the GPCR (G protein-coupled receptor) class16-19. GAGs are expressed by most cell types and are also an integral component of the extracellular matrix20, 21. GAG interactions play a crucial role including in regulating chemokine concentration gradients and receptor activity, which together orchestrate cellular trafficking22.23. Receptor signaling triggers ultrastructural changes that are essential for directed cell migration24,25. The chemokines CXCL1 and CXCL8, members of a subset of seven chemokines that function as agonists for the CXCR2 receptor, orchestrate neutrophil recruitment in response to infection and injury26,27. Sequences reveal two conserved histidines – one located in the N-terminal loop and the second in the 30s loop (Figure 2A). Binding studies have shown that receptor and GAG binding involves only the N-loop histidine and that the 30s loop histidine is remote from the binding interface and cannot be directly involved in binding either to the GAG or the receptor28-32. At this time, little is known regarding the structural features of His or how the imidazole side chain engages its cognate partners in GAG-binding proteins.
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Biochemistry
Figure 2. (A) Sequences of CXCL1 and CXCL8. (B) Ribbon representation of CXCL8 with histidine residues highlighted in red. (C) Schematic showing H-bonding interaction between the His18 side chain and the Lys20 backbone amide proton. (D) NMR evidence for CXCL8 Lys20 backbone amide H-bonding. Overlay of 1H-15N HSQC spectra of CXCL8 at pH 7.5 (black) and pH 3.0 (red) show significant chemical shift change as a function of pH.
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In principle, crystal structures of chemokine complexes should provide insights into which and how histidine imidazole side chain nitrogens engage their cognate partners in the GAG and the receptor. Structures are not available, and even if they were, it is quite challenging to unambiguously identify which of the three forms exists in the bound structure. Most crystal structures lack the resolution to detect protons or to be able to distinguish between nitrogens and carbons. Moreover, H-bonding interactions observed in the crystal structure may not hold in solution phase due to differences in side chain conformation and constraints introduced by crystal packing. On the other hand, NMR chemical shifts and coupling constants of the imidazole ring nitrogens and protons have been shown to be sensitive probes for identifying the ionic and tautomeric states. Indeed, imidazole chemical shifts have been shown quite useful for describing how histidine side chain properties impact protein folding, structure, and function4-10. In this study, we determined the ionic and tautomeric states of histidines in the free and bound forms of CXCL1 and CXCL8 using NMR spectroscopy. Our data indicate that the roles of histidines are quite different between these related proteins. Both histidines in CXCL8 exist in the neutral Nε2 tautomeric state in the free and bound forms. On binding GAG and the receptor, chemical shifts of N-loop histidine alone are perturbed, indicating 30s loop histidine plays no role in binding. In contrast, both histidines in CXCL1 show two distinct sets of peaks characteristic of the charged imidazolium and neutral Nε2 tautomer. In the receptor-bound form, both histidines exist in the Nε2 tautomeric state, whereas both exist exclusively in the imidazolium state in the GAG-bound form. These observations are remarkable, indicating selective binding of one or the other conformer to different binding partners and that the remote histidine adopts the same conformational state as the histidine within the binding interface. We conclude that the structural features and function of histidines can be quite complex, their roles can vary within a protein and between two closely related proteins, and that directly probing the imidazole side chain as described here is essential to understand the molecular basis of histidine function.
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Biochemistry
Material and Methods
Expression and purification of CXCL1 and CXCL8. The recombinant chemokines were expressed in E. coli strain BL21(DE3), and
15
N-labeled protein was produced by growing cells in minimal
medium containing 15NH4Cl as the sole nitrogen source31. Transformed E. coli BL21(DE3) cells were grown in minimal medium containing ampicillin to an A600 of 0.8 and induced with 1 mM isopropyl βD-thiogalactopyranoside
(IPTG) for 8h at 37 °C. Purity and molecular weight of the proteins were
confirmed using analytical high-pressure liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), respectively. The heparin octasaccharide (dp8) was purchased from Iduron (United Kingdom). The main disaccharide unit in dp8 is IdoA,2SGlcNS,6S (∼75%) and shows some variation in sulfation. NMR experiments. NMR spectra were recorded on Bruker 750 and 800 MHz NMR spectrometers at 25 ˚C. The spectra of the CXCL1-dp8 complex were also collected at 40 ˚C. 1H-15N HMQC spectra of the free chemokine and dp8-bound and CXCR2 N-terminal domain-bound complexes were acquired with a 22-ms delay for selective observation of the long-range 1H-15N coupling constant of histidines (2JN-H = 6-12 Hz). The chemical shifts of side chain imidazole nitrogen and proton chemical shifts were assigned as described5. A total of 2048 complex points with a spectral width of 12019 Hz were acquired in the proton dimension, and a total of 256 complex points were acquired in the indirectly detected 15N dimension. The 15N spectral width was 120 ppm centered at 215 ppm. The heparin dp8bound and CXCR2 N-terminal domain-bound complexes were prepared by mixing 200 μM CXCL8 or CXCL1 with heparin dp8 or CXCR2 N-domain in 1:4 and 1:3 molar ratios respectively in 50 mM sodium phosphate, pH 6.0 buffer. The pKa were determined using backbone amide and imidazole side chain chemical shifts of CXCL8 and CXCL1 that were obtained from 1H-15N HSQC and 1H-15N HMQC spectra collected at pH between 2.5 and 8.5.
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Results and Discussion Histidine imidazole nitrogen-attached protons are generally not detected by NMR experiments because of their rapid exchange with the bulk water. However, the non-exchangeable Hδ2 and Hε1 protons (attached to Cδ2 and Cε1, respectively) can be detected via two- or three-bond couplings to the nitrogens in the heteronuclear correlation experiments12,13. Cross-peak intensities reflect the magnitude of the coupling constants, as 2JNH coupling compared to the three-bond 3JNδ1-Hδ2 coupling results in stronger signals, and the latter peak is either weak or often not observed in either the protonated or neutral states (Figure 1). The cross-peak pattern in the HMQC spectrum allows assignment of the ionic and tautomeric states and the imidazole nitrogen and proton (Nδ1, Nε2, Hδ2, and Hε1) chemical shifts. The chemical shifts of ring nitrogens and protons have been reported using model peptides. In the protonated state, Nε2 and Nδ1 have chemical shifts of ~173 and 176 ppm. Nε2 and Nδ1 have chemical shifts of ~164 and 249 ppm in the Nε2H tautomer, and chemical shifts of 249 and 164 ppm in the Nδ1H tautomer. Intermediate values are indicative of fast exchange between the neutral and charged forms. Experimental and theoretical studies have shown Nε2H tautomer is favored by ~5-fold over the Nδ1H tautomer11. To date, NMR studies have characterized histidines in several classes of proteins, from enzymes and glycosidases to those that bind metals and mediate immunity4-10. These studies were mostly focused on characterizing active site histidines, and have shown that chemical shifts are quite sensitive and are composites that reflect their location whether on the surface or buried, their structural environment, and extent of polar, H-bonding, and packing interactions. However, very little is known regarding how histidines mediate macromolecular interactions, and in particular, binding to sulfated polysaccharides such as GAGs. Unlike active site histidines that are buried in the structure, surface histidines mediate macromolecular interactions. In this study, we characterized the structural features of two conserved histidines in two related proteins that interact with GAGs and GPCRs. Role of Histidines in CXCL8 Interactions. The chemical shifts of His18 and His33 imidazole 15N and 1H resonances in CXCL8 were assigned from the 1H-15N HMQC spectrum and previously reported proton chemical shifts33. The chemical shifts and the cross-peak patterns indicated that both His18 and His33 exist in the neutral Nε2 tautomeric state at pH 6.0 (Figure 3). The pKa of the histidines were determined from pH titrations from multiple ring nitrogens and protons, and the data for the side chain protons are shown (Figure 3B). The values of 3.8 for His18 and 4.5 for His33 indicate that the local environment of both histidines is distinctly different compared to a solvent exposed histidine. Low pKa values of
