Hemopexin domain

Fig. 5. Sedimentation equilibrium analysis of hemopexin domain interactions. Mixtures of N- and C-domain were centrifuged to equilibrium at 25 C in the absence (upper panels) and presence (lower panels) of heme. Nonlinear fitting procedures were performed to obtain apparent values, and residuals of the fits are shown in the top portion of each panel (106).

A) as the catalytic-domain structure of HFC. This demonstrates that the absence or presence of the hemopexin domain does not affect the overall structure of the catalytic domain. 

Fig. I. Domain structure of MMPs. The hemopexin domain has a four-bladed propeller configuration. The pre domain is cleaved before exit from the endoplasmic reticulum. MMP-7 lacks the hemopexin domain. MMP-2 and MMP-9 contain fibronectin-binding domains. MT-MMPs (-1, -2, -3, -5) contain a transmembrane and a cytoplasmic domain.

Figure 2 Domain structure of the MMPs 92 kDa gelatinase-A (MMP-2), 72 kDa gelatinase-B (MMP-9), the collagenases (MMP-1, -8, and -13), stromelysin-1 (MMP-3) and matrilysin (MMP-7). Matrilysin is the only known MMP that does not have a C-terminal hemopexin-like domain.

Bode W. A helping hand for collagenases the hemopexin-like domain. Structure 1995 3 527-530. 

Binding of heme by isolated N-domain causes a change in sedimentation coefficient consistent with a more compact conformation and leads to the more avid association with the C-domain (125). Sedimentation equilibrium analysis showed that the Kd decreases from 55 pM to 0.8 pM (Fig. 5) (106). In addition, the calorimetric AH (-1-11 kcal/mol) and AS (-1-65 kcal/mol K) for the heme-N-domain-C-domain interaction and the AH (-3.6 kcal/mol) and AS (-1-8.1 kcal/mol K) derived from van t Hoff analysis of ultracentrifuge data for the interaction in the absence of heme indicate that hydrophobic interactions predominate in the presence of heme and a mix (e.g., hydrophobic and van der Waals forces) drives the interaction in the absence of heme. However, FTIR spectra (Fig. 6) indicate that little change in the secondary structure of domains or intact hemopexin occurs upon heme binding (104). 

The structure of the C-domain of hemopexin was determined first (128). The structure is a four-hladed p-propeller (Fig. 7), the smallest P-propeller known, and serves as the paradigm for the several proteins known to have a pexin domain, including vitronectin (108), and several metalloproteinases (107). The repeats evident in the sequence of hemopexin (99-101), for instance DAAV/F motifs and WD repeat, form a large part of the p-strands of the four blades, which are connected by short loops and a-helices. 

Fig. 7. The crystal structure of the C-domain of hemopexin (PDB accession number IHXN) 128) showed a four-bladed p-propeller structure, which because of sequence similarity was also expected in the N-domain. The high degree of beta structure and limited a-helix content agrees with the earlier FTIR analysis.

Determination of the structure of the entire rabbit heme-hemopexin complex (11) (Fig. 8) clearly revealed the expected complementary structures of the N- and C-domains. More importantly, several fundamental... 

Fig. 8. Crystal structure of heme-hemopexin. The crystal structure of the rabbit mesoheme-hemopexin complex (PDB accession number IQHU) (11) showed heme to be bound in a relatively exposed site between the N- and C-domains with one axial His ligand being contributed by the hinge or linking region between the domains and the other by the C-domain. Also noteworthy is the disposition of the heme with its propionate residues pointing inward and neutralized by positive charges in the binding site.

Fig. 9. EPR spectra of heme-hemopexin and heme-N-domain. X-band EPR spectra at 4 K of ferri-mesoheme-hemopexin (a) and ferri-mesoheme-N-domain (b) are shown. The concentration of both heme complexes was 0.15 mM in 50 50 (v/v) 10 mM sodium phosphate/150 mM NaCl (pH 7.2) glycerol. The g-value scale is noted at the top and the -values observed are noted in each spectrum. Although both complexes are low-spin (some adventitious high-spin iron is present), the differences in g-values indicate nonidentical heme environments in the two complexes (.114).

Fig. 10. H NMR spectra of rabbit hemopexin and domain. The 400 MHz H NMR spectra at 298 K of rabbit apo- and ferri-mesoheme-hemopexin are shown in panels A and B, and the spectra of apo- and mesoheme-N-domain in panels C and D, respectively. The spectra demonstrate that the heme environment in the intact protein is distinct from that in the N-domain. The heme resonances are broadened in the N-domain, consistent with a greater accessibility to solvent 114).

Fig. 11. Absorbance and CD spectra of ferri-, ferro-, and CO-ferro-heme complexes of hemopexin and its domains. Panels A, C, and E show the Soret region absorbance spectra, and panels B, D, and F the corresponding CD spectra. Rabbit hemopexin (panels A and B), N-domain (panels C and D), and N-domain-C-domain (panels E and F) complexes with mesoheme in the ferri- (solid lines), ferro- (dashed lines), and CO-ferro- (dash-double dot lines) states in phosphate-buffered saline, pH 7.4, are presented. The differences between the spectra of hemopexin and the N-domain point to multiple heme binding modes in the protein (139).

Fig. 12. Schematic views of bis-histidyl ferri-, ferro-, and CO-ferro-heme-hemopexin. Unlike myoglobin with one open distal site, heme bound to hemopexin is coordinated to two strong field ligands, either of which a priori may be displaced by CO. This may well produce coupled changes in protein conformation like the Perutz mechanism for 02-binding by hemoglobin (143). The environment of heme bound to hemopexin and to the N-domain may be influenced by changes in the interactions of porphyrin-ring orbitals with those of aromatic residues in the heme binding site upon reduction and subsequent CO binding.

The thermodynamic stability of hemopexin has been examined using DSC (Fig. 13), which showed apo-hemopexin to be a stable protein with a single Tm of 54°C (AH 185 kcal/mol), which increases to 66.5°C (AH 290 kcal/mol) upon binding heme (130). The N-domain of hemopexin is less stable (Tm 52°C, AH 95 kcal/mol) but is even more strikingly stabilized by ferri-heme (T 78°C, AH 370 kcal/mol). The presence of C-domain (Tii 49.5°C, AH 140 kcal/mol) slightly destabilizes heme-N-domain (Tm 75°C, AH 320 kcal/mol) (130), showing another effect of interdomain interactions that may act in heme release. 

Temperature and pH effects on hemopexin, its domains, and the respective heme complexes have also been examined using absorbance and CD spectroscopy, which reflect stability of the heme iron-bis-histidyl coordination of hemopexin and of the conformation of protein, rather than overall thermodynamic unfolding of the protein. Using these spectral methods to follow temperature effects on hemopexin stability yielded results generally comparable to the DSC findings, but also revealed interesting new features (Fig. 14) (N. Shipulina et al., unpublished). Melting experiments showed that apo-hemopexin loses tertiary... 

Fig. 13. DSC of apo-hemopexin and heme-hemopexin. DSC recordings of apo- and heme-hemopexin in 0.05 M sodium phosphate at pH 7.4 and of a mixture of the two are presented. The stabihzation of hemopexin upon heme binding (706) is evident. Other results established an even greater stabihzation of N-domain by heme.

Fig. 14. Effects of temperature on the absorbance of hemopexin and the N-domain of hemopexin. The unfolding of hemopexin and N-domain in 25 mM sodium phosphate, pH 7.4, was examined using absorbance spectroscopy (N. Shipulina et al., unpublished). The second derivative UV absorbance spectra of the protein moieties were used to follow protein unfolding and the Soret and visible region spectra to monitor the integrity of the heme complexes, as done with cytochrome 6502 (166). The ferri-heme complex is more stable than the apo-protein moiety, but the is slightly lower than that assessed by DSC, indicating that changes in conformation occur before thermodynamic unfolding. Reduction causes a large decrease in heme-complex stabihty, which is proposed to be a major factor in heme release from hemopexin by its cell membrane receptor, and addition of 150 mM sodium chloride enhanced the stabihty of ah forms of hemopexin.

An 80- to 90-residue N-terminal propeptide domain contains a cysteine whose -S group binds to the active site zinc, screening it from potential substrates. The central catalytic domain is followed by a hinge region and a C-terminal domain that resembles the serum iron binding and transporting hemopexin.427/436 The mechanism of action is probably similar to that of thermolysin.430... 

Figure 14 (a) The X-ray structure of heme-bound rabbit hemopexin. (b) shows the /S-propeller structure of individual domains. The figure was created using pdb coordinates Iqhu ... 

To date, the crystal structures of 12 different MMPs have been solved. Full structures were obtained for MMP-1 (2CLT), MMP-2 (1CK7), and MMP-7 (IMMP). As for the rest, only the catalytic domains in the presence of different inhibitors were determined. The hemopexin-like domains of MMP-2 (IRTG), MMP-9 (IITV), and MMP-13 (IPEX) were crystallized, and structures were determined separately. Nuclear magnetic resonance (NMR) structures of the catalytic domains of MMP-1 (1AYK), MMP-2 (IHOV), MMP-3 (lUMS), MMP-12 (1YCM), and MMP-13 (lEUB) have also become available. 

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