Heme Binding to Human CLOCK Affects Interactions with the E-Box

Heme binding to human CLOCK affects interactions with the E-box

Samuel L. Freemana, Hanna Kwona, Nicola Portolanob,c, Gary Parkinb,c, Umakhanth Venkatraman Girijab,c,1, Jaswir Basranb,c, Alistair J. Fieldingd, Louise Fairallc,e, Dimitri A. Svistunenkof, Peter C. E. Moodyc,e, John W. R. Schwabec,e, Charalambos P. Kyriacoug, and Emma L. Ravena,2

aSchool of Chemistry, University of Bristol, BS8 1TS Bristol, United Kingdom; bDepartment of Chemistry, University of Leicester, LE1 7RH Leicester, United Kingdom; cLeicester Institute of Structural and Chemical Biology, University of Leicester, LE1 7RH Leicester, United Kingdom; dSchool of Pharmacy and Biomolecular Science, Liverpool John Moores University, Liverpool L3 3AF, United Kingdom; eDepartment of Molecular and Cell Biology, University of Leicester, LE1 7RH Leicester, United Kingdom; fSchool of Biological Sciences, University of Essex, Colchester, Essex CO4 3SQ, United Kingdom; and gDepartment of Genetics and Genome Biology, University of Leicester, LE1 7RH Leicester, United Kingdom

Edited by John T. Groves, Princeton University, Princeton, NJ, and approved August 12, 2019 (received for review March 28, 2019)

The circadian clock is an endogenous time-keeping system that is ligands—such as oxygen (O2), carbon monoxide (CO), and nitric ubiquitous in animals and plants as well as some bacteria. In oxide (NO)—which are themselves established as important mammals, the clock regulates the sleep–wake cycle via 2 basic signaling molecules in cellular control (4). helix–loop–helix PER-ARNT-SIM (bHLH-PAS) domain proteins— The 2 E-box binding proteins—NPAS2 and CLOCK—belong CLOCK and BMAL1. There is emerging evidence to suggest that to the family of transcriptional regulators that contain both basic – – heme affects circadian control, through binding of heme to various helix loop helix (bHLH) domains at the N terminus as well as 2 circadian proteins, but the mechanisms of regulation are largely PAS domains. High sequence identity between these 2 proteins unknown. In this work we examine the interaction of heme with (5, 6) was noted before a direct link between heme and any human CLOCK (hCLOCK). We present a crystal structure for the circadian protein had been established. In this work we present an analysis of heme binding to the PAS-A domain of human PAS-A domain of hCLOCK, and we examine heme binding to the – PAS-A and PAS-B domains. UV-visible and electron paramagnetic CLOCK, we examine the effect of heme on the CLOCK DNA binding interaction, and we present a structure for the PAS-A

resonance spectroscopies are consistent with a bis-histidine li- BIOCHEMISTRY domain. We use this information to put forward ideas on how gated heme species in solution in the oxidized (ferric) PAS-A pro- heme binding might be linked to regulation of circadian control. tein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which Results may give rise to an additional Cys axial ligand at 20K (His/Cys Crystal Structure of hCLOCK PAS-A. A recombinant version (N-terminal coordination). Using DNA binding assays, we demonstrate that His tagged) of the PAS-A domain of hCLOCK (hCLOCK PAS-A, heme disrupts binding of CLOCK to its E-box DNA target. Evidence Fig. 2) was expressed in Escherichia coli. The purified protein is presented for a conformationally mobile protein framework, crystallized as a homodimer in the apo-form (i.e., without heme), CHEMISTRY which is linked to changes in heme ligation and which has the consistent with behavior of the protein during size exclusion capacity to affect binding to the E-box. Within the hCLOCK struc- chromatography (SI Appendix,Fig.S2). tural framework, this would provide a mechanism for heme- dependent transcriptional regulation. Significance

heme | circadian | CLOCK Heme proteins have several well-established functions in biology, from oxygen transport to electron transfer and catalysis. he circadian clock is the internal timekeeping system that But in addition, there is new evidence that heme has a wider Tgenerates a daily rhythm in physiology, biochemistry, and regulatory role in the cell. This includes a role for heme in the behavior of almost all higher organisms and some prokaryotes. – regulation of circadian rhythm. In this work, we have examined In mammalian systems, as in insects, a transcriptional trans- the binding of heme to human CLOCK, one of the positive el- lational feedback loop appears to be at the core of the oscillator ements in the circadian autoregulatory feedback loop. We find (1), and this is coupled to an evolutionarily more primitive – evidence for a conformational mobility within the heme metabolic oscillator (2). The transcriptional translational feed- pocket, and that heme disrupts binding of CLOCK to its E-box back loop has a number of positive and negative components DNA. These results provide a direct link between heme binding that drive interconnected loops that generate the molecular and DNA partner recognition that may form the basis for a clockworks. In mammals, NPAS2 (in the forebrain) and CLOCK mechanism of regulatory control. (in the suprachiasmatic nucleus) form heterodimers with BMAL1 (brain and muscle arnt-like 1) and represent the positive limb of Author contributions: P.C.E.M., J.W.R.S., C.P.K., and E.L.R. designed research; S.L.F., H.K., the feedback loop (Fig. 1). Both dimers bind to the same E-box N.P., G.P., U.V.G., J.B., A.J.F., L.F., and D.A.S. performed research; S.L.F., H.K., N.P., G.P., DNA sequence (CANNTG) to activate expression of the negative U.V.G., J.B., A.J.F., L.F., D.A.S., P.C.E.M., J.W.R.S., C.P.K., and E.L.R. analyzed data; and autoregulators, the 3 Period (Per) and 2 Cryptochrome (Cry)genes E.L.R. wrote the paper with contribution from all authors. (1). PER and CRY, after various posttranslational modifications The authors declare no conflict of interest. that build delays into the loop, then interact with the BMAL1 This article is a PNAS Direct Submission. complexes to negatively regulate their own genes, thereby closing Published under the PNAS license. the loop (Fig. 1 and ref. 1). REV-ERBα, a member of the nuclear Data deposition: The atomic coordinates and structure factors have been deposited in the receptor family, also functions in the cell to repress tran- Protein Data Bank, www.rcsb.org (PDB ID code 6QPJ). scription of BMAL1. 1Present address: Faculty of Health and Life Sciences, De Montfort University, Leicester There is an accumulating body of evidence that indicates that LE1 9BH, United Kingdom. heme is a regulator of the circadian oscillator (3), but the mech- 2To whom correspondence may be addressed. Email: [email protected]. anisms of regulation are entirely unknown. Conceptually, heme This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. binding provides a simple mechanism for signal transduction because 1073/pnas.1905216116/-/DCSupplemental. regulation by heme can be coupled to its ability to bind other

www.pnas.org/cgi/doi/10.1073/pnas.1905216116 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 Fig. 1. Simplified figure of mammalian regulatory clockworks (52). In the positive loop, NPAS2 (dark blue circle) and CLOCK (ditto) form heterodimers with BMAL1 (light blue) which bind to the E-box to activate expression of PER (black) and CRY (orange). PER and CRY heterodimers then interact with the BMAL1 heterodimers to negatively regulate their own genes, thereby closing the loop (1). Other CLOCK-related genes are expressed, including the nuclear receptors REV-ERBα/β (green) and the retinoid receptor orphan receptor (ROR, pink). ALAS, which controls the synthesis (and hence the concentrations) of heme, is also clock regulated. See introductory text for details.

The structure of hCLOCK PAS-A is presented in Fig. 3A. The to the protein and with a 6-coordinate, low-spin heme species. tertiary structure of the overall PAS fold consists of a series of Electron paramagnetic resonance (EPR) spectra are in agree- antiparallel β-sheets forming a hydrophobic core in which a ment with this assignment. The spectrum of heme-bound signaling molecule can bind (7). A number of α-helices are in hCLOCK at 20 K (Fig. 4B) reveal turning points at g1 = 2.93, 1 close proximity which are believed to be responsible for the g2 = 2.28, and g3 ≥ 1.7 typical of a low-spin, ferric (S = /2) heme propagation of structural changes to the rest of the protein or to species (11). These low-spin g-values are consistent with a bis- a partner protein when in a complex. histidine heme coordination. Weak resonances were also ob- eff ∼ eff ∼ The hCLOCK structure has close similarity to that of the O2 served at 4 K at g 6.0 and g 2, which are characteristic of 5 sensor from Rhizobium (FixL, ref. 8) and the heme-regulated high-spin ferric (S = /2) heme (SI Appendix, Fig. S4A). In ad- phosphodiesterase from E. coli (EcDOS) (9, 10), as shown in dition, a shoulder is observed on the central line and attributed = Fig. 3 B and C. A comparison of the heme binding locations in to overlap of a second (minor) low-spin heme species (g1 2.45, = = the 2 structures is informative (SI Appendix, Fig. S3). In the g2 2.28, and g3 1.92). These g-values are similar to those structure of hCLOCK PAS-A, histidine 144, which is implicated previously observed for the isolated PAS-A domain of mouse in heme binding as below, is found on a loop between 2 short PER2 (12). Empirical g-values for heme-bound species are well helices and is oriented toward the β-sheets of the domain in a studied (11). In this case the g-values for the second species in hydrophobic pocket. This hydrophobic core is a feasible heme the EPR are unlike those reported for either His/His or His/Met ligation, and on this basis are assigned as arising from a Cys/His binding pocket, as shown in SI Appendix, Fig. S3. From the − hCLOCK structure, it appears that H144 is a realistic proximal or Cys/OH coordination. = residue for heme and is ideally situated at the mouth of this Heme binds to apo-hCLOCK with a 1:1 stoichiometry (KD ± μ hydrophobic pocket and is surrounded by numerous hydrophobic 4.2 0.25 M; Fig. 4A, Inset). Heme dissociation from hCLOCK residues (Phe121, Leu145, Leu149, Phe157, and Leu198) which PAS-A was measured at 412 nm in the presence of the H64Y/ leave adequate space for a heme molecule. A comparison with V68F mutant of sperm whale myoglobin which has a high affinity FixL shows that the heme in the crystal structure of FixL is lo- for heme (13) (Fig. 4C). A biphasic exponential change was −3 −1 −4 −1 cated in a different location, at the opposite end of this hydro- observed giving rate constants of 8.9 × 10 s and 4.5 × 10 s phobic pocket (SI Appendix, Fig. S3A). for the fast and slow phases, respectively. This biphasic behavior is consistent with the concept of conformational mobility within the Heme Binding to hCLOCK PAS-A. Although heme is not present in heme binding site. The rate constants for heme dissociation are the structure of hCLOCK above, binding of heme to the PAS-A consistent with those derived for other heme regulatory proteins, −3 −1 domain of hCLOCK is clearly evident from UV-visible spectra. such as SOUL and p22HBP (kdiss ∼ 10 s )(14). The spectrum of the oxidized, ferric derivative of hCLOCK PAS- As for the ferric protein above, the ferrous derivative of A(λmax = 413, 533 nm, Fig. 4A) is consistent with heme binding hCLOCK PAS-A (λmax = 425, 533, and 558 nm; Fig. 4D), shows

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Fig. 2. Domain structure in hCLOCK, showing the N-terminal HLH domains and the 2 PAS domains (PAS-A and PAS-B). The protein constructs used in this work are also shown. The vectors used for each construct design are shown in SI Appendix, Fig. S1.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1905216116 Freeman et al. Downloaded by guest on October 1, 2021 AB C

Fig. 3. A comparison of the structure of PAS-A domains. (A) hCLOCK as presented in this work (PDB code 6QPJ). His144 is indicated in red. This is similar to the

equivalent PAS-A domain of mouse CLOCK protein in the CLOCK:BMAL1 complex (53). (B)TheO2 sensor protein from Rhizobium (FixL) (8). (C) The heme-regulated phosphodiesterase from E. coli (EcDOS, refs. 9 and 10). In A, and for mouse CLOCK, the protein crystallizes in the apo-form. In B and C, which both crystallize as the holo-protein, the heme molecule is indicated in red. The Upper figures highlight the binding pocket created by the β-sheets; the Lower structures (which are rotated by 90° about the indicated axis) show the similarity in overall arrangement of the domains. For direct comparison, an overlay of the hCLOCK and FixL BIOCHEMISTRY structures is presented in SI Appendix, Fig. S3.

characteristic heme peaks, which is again consistent with 6- spectrum of the H144A variant (Fig. 4B). The identity of the coordinate, low-spin heme. On the basis of similarities to ferrous proposed Cys ligand seen only at low temperature in the EPR NPAS2 (λmax = 423, 530, and 558 nm) (15), which is assigned as a (above) was not established. The most likely candidate was His/His ligated heme species (16), we assign hCLOCK PAS-A as considered to be Cys195 which points toward the heme pocket,

the same. Ferrous hCLOCK binds both CO (λmax = 421, 540, and but the EPR spectrum of the C195A variant (which corresponds CHEMISTRY 570 nm; Fig. 4D) and NO (λmax = 421, 531, and 556 nm; Fig. 4D), to Cys170 in NPAS2 and has been suggested as a ligand) (20) was with a presumed His/CO ligation. In kinetic experiments mea- unchanged from that of the wild-type protein (Fig. 4B). The UV- suring CO binding to hCLOCK PAS-A, pseudo first-order rate visible spectrum of the C195A variant is also very similar to the constants (kobs) showed a nonlinear dependence on the concen- wild type (SI Appendix, Fig. S4B) and thiol modification of apo- tration of CO, but were linearly dependent on the protein con- hCLOCK with DTNB (to modify all Cys residues) followed by centration (Fig. 4E). This kinetic behavior is consistent with a reconstitution with heme did not lead to a change in the UV- dynamic equilibrium of the protein structure (P) prior to CO visible spectrum. Mutation of the adjacent Cys194 residue gave a λ = binding (17), presumably leading to loss of one of the axial His similar heme-bound spectrum ( max 414 nm) and was presumed ligands, to bind heme normally in solution by virtue of the His144 ligand. Together, we interpret this information to mean that the Cys- k +CO, k ligated species is only present at low (cryogenic) temperatures. 1 * !2 P ⇄ P P − CO, [1] We also expressed a PAS-B fragment of hCLOCK (Fig. 2 and − k−1 CO, k−2 SI Appendix, Fig. S1), which was found to bind heme in the ferric form (λmax = 416 and 536 nm) (SI Appendix, Fig. S6) but with and in the case where CO binding is irreversible (k-2<< k1k2/k-1), marginally weaker affinity (KD = 13.3 ± 0.97 μM) (SI Appendix, a hyperbolic dependence of kobs on CO concentration is observed, Fig. S6) than the PAS-A form of hCLOCK. The PAS-B domain = ½ =ð + ½ Þ [2] of NPAS2 is also reported as binding heme more weakly than the kobs k1k2 CO k−1 k2 CO . corresponding PAS-A domain (21) but the functional conse- quences, if any, of this difference are not known because the role The addition of oxygen to ferrous protein under anaerobic of PAS-B is not yet established. The ferrous (λmax = 424, 532, conditions gave no evidence of a ferrous-oxy intermediate—this and 559 nm) (SI Appendix, Fig. S6) and CO-bound forms (λmax = might be linked to the low reduction potential of the heme in 421, 541, and 569 nm) (SI Appendix, Fig. S6) also form normally hCLOCK PAS-A, which has been previously determined (18, 19) in PAS-B. Like the hCLOCK PAS-A domain, these spectra are as −111 mV vs. NHE. indicative of a 6-coordinate heme binding interaction but this Based on a sequence alignment with NPAS2 (SI Appendix, Fig. was not examined further in this work. S5), we identified His144 as a possible ligand to the heme in hCLOCK (equivalent to the axial His119 ligand in NPAS2). The Heme Affects Binding to the E-Box. To assess the functional effect corresponding spectra of the ferric H144A mutant of hCLOCK of heme on DNA binding, we expressed a construct of hCLOCK PAS-A in the presence of heme showed a broad, strongly blue- PAS-A containing an N-terminal HLH domain (Fig. 2 and SI shifted Soret band (λmax = 374 nm) which is different from that Appendix, Fig. S7A). The hCLOCK HLH/PAS-A construct was of the ferric wild-type protein and which is not consistent with a demonstrated to bind to E-box DNA (Fig. 5A); a plot of the bona fide heme protein (Fig. 4F). On this basis, we assign His144 relative band intensity versus [hCLOCK HLH/PAS-A] gave a as one of the heme ligands in hCLOCK. EPR spectra confirm KD = 6.3 μM, which is similar to that determined (KD = 9 μM) this assignment as the g1 = 2.93 resonance is missing in the for a different construct of CLOCK containing only the HLH

Freeman et al. PNAS Latest Articles | 3of6 Downloaded by guest on October 1, 2021 logical and potentially versatile. Heme is redox active and, thus, A B responds to redox changes within the cell by modulating its oxidation state. Concentrations of heme in the cell are controlled by the balance of heme biosynthesis (to increase heme concentrations) and heme oxygenase activity (which degrades heme and produces CO as a byproduct). Cell-signaling gases (O2, NO, and CO) all bind to heme, which creates a possible mechanism of regulatory control. CO is itself produced by degradation of heme by the O2-dependent heme oxygenase enzyme, and NO is produced by the O2-dependent NO synthase enzyme. When one C D considers this in the context of circadian control, this means that the redox state of the cell, the oxidation state of heme, the balance between heme synthesis and degradation, and O2/NO/CO concentrations may all be interconnected. This is summarized in outline form in Fig. 1. This interconnectivity could form the basis for a powerful heme-dependent circadian control mechanism that reflects metabolic activity. The fact that transcription of ALAS1, the first enzyme in the heme synthesis pathway, and other genes involved in heme catabolism (such as heme oxygenase) and heme EF transport (23–29) are also clock regulated is also significant as a means of connecting heme synthesis, heme degradation and CO formation to circadian events. Indeed, it has previously been shown that heme-related genes in mouse are also implicated in the light resetting of the clock either as cause or effect (30). In the case of particular circadian proteins, several—including REV-ERB, PER2, NPAS2—are shown to bind heme (12, 15, 20, 21, 31–40) and complex formation with partner proteins can be affected by heme or CO binding (34, 41, 42). And there are precedents for a circadian control mechanism that occurs through binding to PAS domain proteins, because of other regulatory PAS Fig. 4. (A) UV-visible absorption spectra of the ferric heme/CLOCK PAS-A domain proteins that are known to bind heme [for example FixL complex. (Inset)ShowsΔAbs at 415 nm (obtained from difference spectra at in the histidine kinase family, the phosphodiesterases EcDOS and various heme concentrations) as a function of heme concentration and fitted to a PDEA1 (43) and Burkholderia xenovorans RcoM (16)]. 1:1 binding event. (B) The 9 GHz EPR spectra of ferric heme bound to hCLOCK PAS-A (Top), the H144A variant of PAS-A (Middle), and the C195A variant of PAS- A(Bottom) in 50 mM Tris/HCl buffer at pH 7.0. (C) Absorbance change at 412 nm for the dissociation of heme from hCLOCK PAS-A (3 to 4 μM) on mixing with apo- A B H64Y/V68F myoglobin (80 μM). Data are fitted to a double exponential process. (D) Spectrum of the ferrous (solid line), CO-bound (dotted line), and NO-bound (dashed line) derivatives of heme-bound hCLOCK PAS-A. (E) Observed rate constants for the association of CO with hCLOCK PAS-A, shown as a function of [CO] (Top)and[hCLOCKPAS-A](Bottom). (F) Spectrum of the ferric heme/CLOCK PAS- A H144A complex (solid line) compared to free ferric heme (dotted line). This spectrum resembles that of the related His335 variant of NPAS2 PAS-B (where H335 is assigned as 1 of the heme ligands) (21).

domain (22). Control experiments (Fig. 5B) with the corresponding HLH/PAS-A construct of NPAS2 (SI Appendix, Figs. S7B and S8) similarly demonstrated binding of NPAS2 to E-box = μ (KD 11 M, Fig. 5B). These HLH/PAS-A proteins are pre- C D sumed to bind to DNA as homodimers, as HLH domains are unable to form a specific interaction with E-box DNA in the monomeric form. Addition of heme inhibits DNA binding to the HLH domain of hCLOCK, as evidenced by the shift in the DNA band in electrophoretic mobility shift assays (EMSAs) (Fig. 5C); the same effect is observed for NPAS2 (Fig. 5D). From the intensities of the DNA–protein and the the free DNA bands, an IC50 of 7.4 μM was derived for the inhibitory effect of heme on DNA binding to hCLOCK; this value is in the same range as the KD for heme binding to the PAS-A domain. Discussion The Role of Heme in Circadian Regulation. In addition to known roles as a prosthetic group in a multitude of heme proteins and enzymes, there is now emerging evidence that heme has a wider Fig. 5. Electrophoretic mobility shift assays showing DNA binding to the role to play in biology. A regulatory mechanism in cells that is HLH/PAS-A domains of hCLOCK (A and C) and NPAS2 (B and D) in the ab- linked to heme binding is, to our way of thinking, chemically sence (A and B) and presence (C and D) of heme.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1905216116 Freeman et al. Downloaded by guest on October 1, 2021 Structure and Heme Ligation in hCLOCK. We have presented a structure for the PAS-A domain of hCLOCK. Heme is not bound to hCLOCK in this structure, although there is con- vincing evidence for noncovalent heme binding in the UV- visible and EPR spectra, as well as from the kinetic data. The absence of heme in our crystal structure does not imply a lack of specific heme binding—it may be that the particular conformation in which hCLOCK PAS-A crystallizes is not com- patible with heme binding correctly in the cavity and thus heme is excluded during crystal formation. An absence of heme in the structure of a presumed heme binding protein has also been noted in the periplasmic heme transporter protein BhuT (44); this has been attributed to structural flexibility of the transporter complex. Heme binds relatively weakly to the PAS-A domain of Fig. 6. The hCLOCK PAS-A crystal structure as presented in this work (in hCLOCK, and weak heme binding is a feature of a number of pale blue, PDB code 6QPJ) superimposed onto a homology model of the regulatory heme binding proteins (20). The heme is proposed to PAS-A domain of NPAS2 (in light gray). Cys195 (equivalent to Cys171 in reside at the mouth of a hydrophobic cavity comprising residues NPAS2, overlaid in gray) and Cys194 (equivalent to Tyr169 in NPAS2, not Phe121, Leu145, Leu149, Phe157, and Leu198 (SI Appendix, Fig. shown) in hCLOCK are shown in yellow; Cys250 in hCLOCK is also shown. S3). This is plausible in the sense that FixL also binds heme in a His144 in hCLOCK is highlighted in red, overlaid with His119 in NPAS2 (in hydrophobic pocket (residues Val188, Met192, Tyr203, and gray). The homology model was produced using SWISS-MODEL (Biozentrum, Ile204) that helps to orient the heme and the His ligand (His200) University of Basel) using the PAS-A domain of the mouse CLOCK structure in the correct location (8). Our comparison of the structures (SI (PDB code 4F3L) (54). The figure was created using Pymol (55). Appendix, Fig. S3) indicates that the heme binds at different locations in hCLOCK and FixL—both locations appear to be viable but the biological relevance of these differences is not heme in the crystal structure, and the required loss of the axial yet known. His ligand on CO binding to the ferrous form. This type of Fromtheavailablespectroscopic data for hCLOCK we assign protein flexibility has been noted in other regulatory proteins the heme binding interaction in ferric hCLOCK as histidine ligated (47) as well as in heme-based sensors (48), and could be a at room temperature in solution. Data for the His144 variant common feature of the way that these regulatory heme proteins BIOCHEMISTRY strongly implicate this residue as one of the heme ligands, and operate. Further structural information will be important in His144 is essential for heme binding. The hCLOCK protein ap- resolving these questions. pears to contain 6-coordinated (His/His) heme as evidenced by EPR, but appears able to adopt other distinct states with the axial Effect of Heme on DNA Binding and Functional Implications. Our data ligand(s) being labile. The identity of the second histidine ligand is demonstrate that heme inhibits binding of hCLOCK to DNA. not known. There is evidence for a 5-coordinated ferric high-spin This provides evidence for an influence of heme on the DNA

heme species from the EPR, again consistent with axial ligand la- binding ability of CLOCK in cells. How might this work and how CHEMISTRY bility (but His144 is presumed not to be labile, as it is essential for might it affect signal transduction? We offer the following com- heme binding). These data are in agreement with those for NPAS2, ments. If the proteins are conformationally mobile, for which which assign the equivalent residue (His119) as an axial ligand (20) there is evidence (as above), then it is plausible that heme binding (SI Appendix,Fig.S5). There is evidence from EPR only (empirical to the PAS domain causes structural rearrangements that are interpretation of the EPR g-values) for an additional Cys ligand to transmitted and affect the interaction with the E-box. Based on the heme in hCLOCK at 20 K. Cys ligation has been suggested in the weak heme binding affinity, it is feasible that the heme binding other heme regulatory proteins (16), but the spectroscopic evidence event is itself reversible; reversible loss of heme has been noted in for it in the case of NPAS2 (where Cys170 has been proposed as a other systems recently (49) and could contribute to the regulatory ligand, equivalent to Cys195 in hCLOCK) (SI Appendix,Fig.S5), control. The PAS domains that have been studied appear to ini- and particularly in mouse PER2, is not totally conclusive. In the tiate signaling from the central β-sheet region of the structure case of NPAS2, axial ligation might be affected by the presence of (50); the signal is then often propagated to the Cα helix of the PAS the N-terminal HLH domain (12, 20, 45). For hCLOCK, there are domain. His144, which binds to the heme in hCLOCK, is located 3 Cys residues in the sequence and one of these (Cys250) is at >20 Å on the Cα helix, and conformational changes linked to heme from His144. Cys195 points toward the pocket, but is still at >10 Å binding in this proposed signaling region would therefore provide from the His144 ligand. The adjacent Cys194 residue is exposed a convenient mechanism for signal transduction. Binding of sig- and pointing away from the proposed heme pocket. A homology naling gases to the heme (such as CO) might also lead to structural model of hCLOCK and NPAS2 (Fig. 6) indicates an overlay of the rearrangements in the heme pocket, and there is evidence for this His144/His119 and Cys195/Cys170 residues. From the evidence in the related FixL sensor protein (51). How these signals are available so far, our conclusions are that the axial ligand iden- further propagated is not yet known. tities in hCLOCK—and, by analogy, in other related regulatory proteins—are not as straightforwardly defined as in other well-known Materials and Methods heme proteins, such as myoglobin (His/H2O), cytochrome b5 (His/ Detailed methods on the following subjects are available in SI Appendix, SI His), cytochrome c peroxidase (His/H2O), or cytochrome P450 Materials and Methods: cloning and mutagenesis, expression and purifica- (Cys/H2O) which have more rigidly coordinated heme ligands. tion, electronic spectroscopy, crystallization, kinetic measurements, and EMSAs. Instead, we envisage these regulatory heme proteins as being — much more fluid in their heme binding and thus able to ACKNOWLEDGMENTS. We are grateful to the late Dr. X. Yang (University change their axial ligation in response to different stimuli, which of Leicester PROTEX cloning facility) for providing the expression con- could be binding to a protein partner, to DNA, to another heme structs used for this work and Dr. J. Olson (Rice University) for providing us ligand (such as CO), or to a change in oxidation state (16). This with apo-H64Y/V68F SwMb. We thank Toru Shimizu for sharing unpub- would require a degree of flexibility in the heme pocket which, lished spectra on NPAS2. This work was funded by the Biotechnology and Biological Sciences Research Council (grant BB/L006626/1 to E.L.R.). We in the case of the CLOCK protein, is consistent with the fact thank the National Engineering and Physical Sciences Research Council that PAS domains are known to be conformationally mobile (46). EPR service and facility for support (NS/A000055/1) and Louise Bird and We have good evidence for conformational mobility of hCLOCK Raymond Owens (Oxford Protein Production Facility, Harwell) for help in our observations, namely the weak heme binding, the absence of with cloning.

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