The N-terminal domain of human holocarboxylase synthetase facilitates biotinylation via direct interaction with the substrate protein

2.1. Limited proteolysis of full-length hHCS 

FL-HCS was processed by incubation with several proteases at a 100:1 (w/w) substrate-to-enzyme ratio for 1h at 4°C. For the analytical sample, the digestion was stopped by heating in SDS loading buffer for 5min at 95°C. The digestion products were resolved in a reducing and denaturing 12% polyacrylamide gel.

2.2. NMR spectroscopy 

The NMR measurements were performed at 20°C on either 0.5mM 15N-labeled apo-hACC75 or 0.5mM 15N-labeled holo-hACC75, both of which contained 50mM HEPES (pH 7.3), 150mM NaCl, 1mM DTT and 10% D2O. For the chemical shift perturbation (CSP) experiments, we recorded the NMR spectra at 20°C using samples that contained 0.25mM 15N-labeled apo-hACC75 and either 50μM 160-HCS or 0.25mM NTD–HCS, using the same buffer conditions. The combined amide CSP was calculated as Δδ=[(ΔδN/5)2+(ΔδH)2]1/2, where ΔδN and ΔδH are the shifts in the 15N and 1H signals, respectively [20]. The NMR spectra of NTD–HCS, NTD–HCS with hACC75 and 161-HCS were recorded at room temperature using the same buffer conditions. We used a 5:1 molar ratio of 15N-labeled NTD–HCS to the non-labeled protein (hACC75 or 161-HCS). The NMR data were recorded on an Avance 500 spectrometer (Bruker) equipped with a cryogenic probe, and the data were processed with NMRPipe and analyzed using Sparky 3.114.

2.3. Circular dichroism 

The circular dichroism spectra were acquired using a J-710 spectropolarimeter (Jasco). The NTD–HCS sample (30μM) was measured using a 1mm path in 20mM Tris (pH 7.5) and 50mM NaCl. Data were acquired using a scan rate of 0.2nm/s, and the data from five scans were averaged. The baseline was corrected using a spectrum that was taken of the same buffer without protein. The data were converted to the mean residue ellipticities [θ] in degcm2/dmol using the equation=(θobs/10lc)/r, where θobs is the ellipticity measured in millidegree, l is the optical path length (cm), c is the concentration of protein (M) and r is the number of residues. The composition of the secondary structure elements of the protein was deconvoluted from the CD spectrum (195–260nm) using the neural network-based software CDNN (version 2.1) [21].

2.4. In vitro biotinylation assay 

The biotinylation of hACC75 was detected quantitatively using avidin blots. The reactions, containing 80μM apo-hACC75, 50mM Tris–HCl (pH 8.0), 3mM ATP, 5.5mM MgCl2, 1mM DTT and 500μM biotin, were carried out at 30°C. The reaction was initiated by the addition of purified full-HCS or 161-HCS to a final concentration of 15nM. When the mixture of 161-HCS and NTD–HCS was used for biotinylation, the mixture was incubated at 4°C overnight. Samples of the reaction mixture were taken at constant time intervals and resolved using an 18% polyacrylamide gel, before being transferred to a PVDF membrane. The non-specific sites on the membrane were blocked using 1% BSA and then incubated with streptavidin-HRP (1:1000) for 1h at room temperature. The membrane was developed using AEC/H2O2. The biotinylated proteins were analyzed using ChemiDoc XRS (Bio-Rad) and Quantity One software.

3.1. Structural characterization of the N-terminal region of hHCS 

Purified intact hHCS was subjected to limited proteolysis with several proteases to define the structural boundaries of the N-terminal domain within the enzyme. The protein was treated with subtilisin, proteinase K and chymotrypsin, and the products were analyzed using SDS–PAGE (Supplementary Fig. S1A). All three proteases generated a fragment of about 65kDa, which contained the C terminus, as identified by Western blotting against the His-tag antibody (data not shown). N-terminal sequencing of these products revealed that the cleavage occurred between 126E and 127N for subtilisin, between 127N and 128I for proteinase K, and between 100H and 101L for chymotrypsin (Supplementary Fig. S1B). This result indicates that the protein contains a protease-sensitive region between residues 100 and 130.

Polyak et al. [17] have demonstrated the sensitivity of the enzyme to protease cleavage using yeast BPL, and they identified a protease sensitive site between Lys 240–Asn 260 in yeast BPL. A flexible region, located between residues 240–260, divides yeast BPL into a 27kDa N-terminal domain and a 50kDa C-terminal domain. Similarly, our limited proteolysis study of purified full-length hHCS has provided information that has identified an N-terminal domain similar to that of yeast BPL. In addition, based on analyses of the secondary structure (http://bioinf.cs.ucl.ac.uk/psipred and http://ffas.burnham.org/XtalPred), the residues from 130 to 160 are predicted to form a random structure, whereas the residues from 160 to the C-terminus are predicted to form a folded structure. Together with the limited proteolysis results, the secondary structure prediction suggests that the N-terminal residues (1–160) form a domain that is independent from the remaining part of human HCS. Therefore, in this study, we designed an N-terminally truncated HCS starting at Ala 161 (161-HCS) and we assigned the N-terminal domain as the fragment from Met 1 to Lys 160 (NTD–HCS).

To characterize the structure of the N-terminal domain of hHCS, we expressed and purified the N-terminal 160 residues (NTD–HCS) in an E. coli system. The amide protons in the heteronuclear single quantum coherence (HSQC) spectrum of 15N-labeled NTD–HCS appeared in the region from 8.0–8.5ppm, and this is characteristic of a random coiled structure (Supplementary Fig. S2). The CD spectrum showed a strong negative peak below 200nm, which is characteristic of a random coil configuration, and a negative peak around 222nm, suggesting some content of α-helicity (Supplementary Fig. S3). Using the CDNN program [21], we estimated the secondary structure of the NTD–HCS to be mainly random coil, with some α-helical structure, consistent with the result of secondary structure prediction mentioned above. NTD–HCS has a large number of charged residues (Lys, Arg, Glu, and Asp) and there are 12 proline residues that are distributed randomly in the sequence. Thus, we assume the N-terminal domain to have mainly a random coil structure with some α-helical content.

3.2. The activity of full-length and N-terminally truncated hHCS 

When the apo-biotinoyl domain is biotinylated to form the holo-protein, the chemical shifts of the residues that are located near Lys 929 are changed significantly (Fig. 1A). To compare with the activity of hHCS, we analyzed the biotinylation of the human ACC2 biotinoyl domain (hACC75) by NMR in the presence of either full-length hHCS (FL-HCS) or N-terminally truncated hHCS (161-HCS) containing the catalytic domain. After 5h reaction with FL-HCS, the 1H–15N HSQC spectrum of hACC75 showed full conversion of the biotinoyl domain to its holo-form (Fig. 1B). However, using 161-HCS, the signals from apo-hACC75 still showed substantial intensities (Fig. 1C), indicating that the enzymatic activity of 161-HCS was decreased significantly by the N-terminal truncation. Even after 12h, although the intensities were greatly reduced, the peaks corresponding to apo-hACC75 remained, showing that the NMR signals from the apo-protein coexisted with those of the holo-protein (Fig. 1D). These results indicate that the enzymatic activity of 161-HCS was significantly reduced by the truncation of the 160 N-terminal residues.

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Fig. 1. Activity monitoring using NMR. (A) Overlay of the 2D 15N–1H HSQC spectra for 15N-labeled apo- and holo-hACC75. The cross peaks for the apo- and holo-proteins are shown in black and red, respectively. Chemical shift changes were observed for residues near Lys 929. The blue boxed region is magnified and shown in (B), (C) and (D). Overlay of the 2D 15N–1H HSQC spectra before and 5 hours after biotinylation using FL-HCS (B) and 161-HCS (C) at 30°C. (D) Overlay of the 2D 15N–1H HSQC spectra before and 12 hours after biotinylation using 161-HCS. The black cross peak represents the protein before biotinylation, when only the apo-protein exists. The red cross peak represents the protein after biotinylation.

When the biotinylation reactions were monitored using avidin blots, similar reaction rates were observed. hACC75 was biotinylated by FL-HCS, yielding saturation bands on an avidin blot between 3 and 6h (Fig. 2A). However, when 161-HCS was used in the biotinylation reaction, the band corresponding to the biotinylated contents did not reach saturation at 6h after the start of the reaction. This result also indicates that removal of the N-terminal domain reduces hHCS activity significantly.

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Fig. 2. Avidin blots of holo-hACC75 at 30°C. The bands show the amount of biotinylated protein sampled at various time increments following the addition of HCS. Lane 1, before biotinylation (no ligase added); lanes 2–4 correspond to 1, 3 and 6h after the addition of ligase; lane 5, identical amounts of holo-protein used as a control (completely biotinylated hACC75). Biotinylation using FL-HCS, 161-HCS and NTD–HCS are shown in (A), (B) and (C), respectively. The enzyme mixtures, at 1:1 and 1:2 molar ratios of 161-HCS to NTD, were also used for biotinylation and are shown in (D) and (E), respectively.

Similar to our findings, N-terminally truncated BPL in yeast showed a 3500-fold reduction in activity [17], and the authors proposed that the presence of the N-terminal domain is necessary to produce a functional enzyme. They assumed that in the absence of the N-terminal domain, the conformational changes that are associated with substrate binding and are necessary for enzymatic activity may occur at a slower rate than in the presence of the N-terminal domain. Therefore, the overall activity of the protein will be affected [17]. Furthermore, although a shorter deletion of 79 amino acids results in a fully active hHCS, the enzymes that began at Leu 165 or Leu 166 did not show any activity in the in vitro assay [18]. Our findings that the absence of the N-terminal domain causes a significant defect in the activity of hHCS are consistent with these results.

3.3. The effect of NTD–HCS on the biotinylation activity 

As mentioned above, the N-terminally truncated version of hHCS has defective activity. To investigate the potential role of NTD–HCS on enzyme activity, we carried out the biotinylation using 161-HCS in the presence of NTD–HCS. The avidin blotting for the biotinylation reaction using purified NTD–HCS did not show any bands, implying that, as expected, it is unable to biotinylate the biotin acceptor domain of human ACC2, hACC75 (Fig. 2C). To our surprise, however, the addition of NTD–HCS to the reaction mixture containing 161-HCS at a 1:1 molar ratio showed full biotinylation activity in vitro, as does full-length hHCS (Fig. 2D and E). This result indicates that the NTD–HCS has a critical functional role that can rescue the N-terminal truncation of hHCS with respect to the catalytic reaction with 161-HCS.

Several studies of human HCS have proposed that the N-terminal domain contributes to biotinylation and that it may affect acceptor substrate recognition. Previously, it was shown that various N-terminal deletion constructs of HCS that were prepared by expression of exonuclease digestion products of the gene had different abilities to biotinylate substrates (the biotinoyl domain of propionyl-CoA carboxylase and E. coli BCCP). This implied that disruption of the N-terminal domain interferes with the biotin transfer reaction [18]. Furthermore, yeast-two-hybrid (Y2H) assays suggested that this N-terminal region (1M-446F) may be involved in substrate recognition [11]. More recently, Ingaramo and Beckett demonstrated that the N-terminus (1M-57G) affects the kinetics [5]. Based on our results, and considering that the NTD–HCS can rescue the catalytic function of the N-terminal truncation (even though it is not attached to the main body of the enzyme), it is clear that NTD–HCS plays a cooperative role in the catalytic function with the catalytic domain. We suggested that there is a functional interaction with either the biotin acceptor protein or the catalytic domain.

3.4. The interaction of NTD–HCS with the substrate and the core domain of hHCS 

To investigate the interaction of NTD–HCS with the remaining part of hHCS and/or the substrate biotinoyl domain, we performed NMR CSPs experiments in the presence of 161-HCS or hACC75. The addition of 161-HCS (including the catalytic domain) to NTD–HCS induced weak chemical shift changes in the 1H–15N HSQC spectra, and several signals were broadened (Fig. 3B). This result indicates that NTD–HCS interacts directly with 161-HCS with weak affinity. CSPs were also observed when titrating hACC75 with NTD–HCS (Fig. 3C). These results indicated that NTD–HCS can interact directly with both 161-HCS and hACC75. The chemical shift changes of representative signals are shown in the enlarged spectra that are shown in Fig. 3D, indicating the concentration dependency of hACC75. Interestingly, when hACC75 was added to the N-terminal domain, many cross peaks became more intense rather than broadened (Fig. 3C). This result implies that the chemical exchange rate of NTD–HCS in solution was affected by the titration of hACC75, resulting in the appearance of new signals and higher intensities of the NMR signals (possibly due to faster or slower conformational exchange). We suggested that when NTD–HCS binds to the substrate, some conformational change in NTD–HCS occurs.

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Fig. 3. Chemical shift perturbation in the NTD–HCS system. (A) The 2D 15N–1H HSQC spectrum of NTD–HCS (pH 7.3, Temp=25°C, 10% D2O). (B) Overlay of the 15N–1H HSQC spectra for the 15N-labeled NTD–HCS before and after the addition of 161-HCS. Black represents NTD–HCS and magenta represents the NTD–HCS in the presence of 161-HCS. (C) Overlay of the 15N–1H HSQC spectra for 15N-labeled NTD–HCS before and after addition of hACC75. Black represents NTD–HCS and red represents NTD–HCS in the presence of hACC75. (D) The magnified region of the peaks that are numbered in (C). Control NTD–HCS is shown in black, and the spectra obtained using molar ratios of 5:1 and 1:1 of NTD–HCS to hACC75 are shown in red and blue, respectively. Each color bar indicates the respective range of the chemical shift change.

3.5. Recognition of the hACC75-binding surface by NTD–HCS 

To understand how NTD–HCS interacts with the biotin acceptor protein (hACC75), we conducted CSP experiments, comparing NTD–HCS and the N-terminally truncated enzyme (161-HCS). We analyzed changes in the binding site of hACC75 that occurred on the addition of NTD–HCS and 161-HCS. Fig. 4 presents the CSPs together with the residue number and the mapping of the binding surfaces of hACC75 in each experiment. The residues on the surface of hACC75 that were perturbed due to the addition of 161-HCS were essentially the same as those that were perturbed by the addition of FL-HCS (data not shown). The results indicate the key residues that are involved in the binding, including the MKM motif and glutamic acid residues. However, the addition of NTD–HCS induced the perturbation of NMR signals that are associated with charged residues on the surface of hACC75, and this is distinct from the result with 161-HCS. It is notable that NTD–HCS can interact with the different surfaces of hACC75 and 161-HCS, suggesting a cooperative interaction with 161-HCS. Together with the result that indicates the recovery of the biotinylation activity from the N-terminal truncation by the addition of NTD–HCS, this result implies that NTD–HCS may recruit the substrate to facilitate the biotinylation reaction.

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Fig. 4. The analysis of the chemical shift changes of hACC75. (A) The plot represents the combined amide CSP differences between the HSQC spectra of apo hACC75 in the presence and absence of 160-HCS. (B) The CSP differences between apo-hACC75 in the presence and absence of NTD–HCS. (C) and (D) are surface representations of hACC75, colored according to the chemical shift change induced following the addition of 161-HCS (C) and NTD–HCS (D).

In conclusion, we have determined the structure of the N-terminal domain of hHCS by limited proteolysis and demonstrated that this region interacts with both the core body of hHCS that contains the catalytic domain and the biotin acceptor. Furthermore, the CSP experiments suggest that the charge-charge interaction contributes to the recognition of the biotin acceptor protein by the N-terminal domain of hHCS. These results may provide structural insights into the substrate recognition by the N-terminal region of hHCS.