The C domain

The complementation experiments in which the A domain of a class 111 E-II is used as the phosphoryl group donor to the B domain of a second E-II molecule with either the same or different sugar specificity, while both are fixed in a membrane matrix, raises some intriguing issues about the association state of these proteins and the kinetics of their interactions. Do E-IIs form stable homologous complexes in the membranes If so, is it necessary to postulate the formation of stable heterologous complexes to explain, for example, the phosphorylation of the B domain of E. coli 11° by the A domain of ll , or can the data be explained by assuming a 

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Fig. 4. Domain complementation schemes. (A) A domain complementation. The H554A site-directed mutant is inactive in P-enolpyruvate-dependent mannitol phosphorylation because it cannot accept a phosphoryl group from P-FIpr. The measure of A domain activity is its ability to restore mannitol phosphorylation activity to this mutant. A domain activity in the AB subcloned protein can also be measured. (B) B domain complementation. The C384S site-directed mutant is inactive in P-enolpyruvate-dependent mannitol phosphorylation because it cannot pass the phosphoryl group from H554 on its own A domain to mannitol. The measure of B domain activity is its ability to restore mannitol phosphorylation activity to this mutant. B domain activity in the AB subcloned protein can also be measured. (C) C domain complementation. The activity of the C domain is measured by complementation with the purified AB domain.

At 293 K two calcium ions bind cooperatively at the C-domain, with logK = 7.3, 7.6. 

In another recent example, Hashimoto reported photoaffinity experiments on retinoic acid receptors (RAR). Retinoic acid plays a critical role in cell proliferation and differentiation. RARs belong to the superfamily of nuclear/ thyroid hormone receptors. They consist of six transmembrane domains (A-F) which is a general feature of these receptors. The A/B domains have an autonomous transactivation function while the C-domain contains the Zn-finger, which binds to DNA. The large E-domain participates in ligand binding, dimerization, and ligand dependent transactivation. Finally, D- and F-domains help the orientation and stabilization of the E-domain. 

Fig. 10.4. HsIVU activation mechanism. (A) Stereo view (C -trace) of the superposition of the C-domain of an HsIU subunit (red) from the original E. coli HsIVU complex onto that of the H. influenzae HsIU subunit (green) from its complex Two HsIV subunits (pink and blue) from the H. influenzae complex are also shown to illustrate the binding of the C-terminal segment of H. influenzae HsIU to the pocket between the HsIV subunits (indicated also by a...

Angiotensin converting enzyme (ACE) is an important enzyme for the regulation of blood pressure, ft exists in two forms the somatic form has 1277 amino acids, while the sperm cell form has 701 amino acids. The somatic form consists of two domains the carboxy-terminal (C) domain and the amino-terminal (N) domain. The sperm cell form consists of only the C domain. Studies have shown that the C domain is the dominant angiotensin converting site for controlling blood pressure and cardiovascular functions. 

Figure 8 Crystal structure of the C-A-T-TE SrfA-C termination module from Bacillus subtilis. The C domain is shown in gray, the A domain in yellow and blue, the T domain can be seen in red, the TE domain in green, and the C-terminal tag is shown in orange.

The structurally related myxochromides Aj.j are cyclic hexapeptides produced by several Myxococcus species. These examples contain a proline residue, which is not present in myxochromides Si 3, as the fourth amino acid in their peptide core. The NRPSs responsible for myxochromides A and S biosynthesis have exacdy the same module and domain organization thus, the fourth module of the myxochromide S synthetase must be skipped to account for the natural product. Biochemical experiments revealed that the A domain of this module activates L-proline, but the adjacent PCP domain cannot be phosphopantetheinylated by a PPTase. These results suggest that the C domain of module 5 reacts directly with the tripeptide intermediate bound to the PCP domain of module 3 in myxochromide S biosynthesis. A similar example of domain skipping has been noted in the biosynthesis of the mannopeptimycins. ... 

Another set of unusual C domains include those that catalyze the formation of more than one amide bond on an acceptor substrate containing multiple amine moieties. For example, the C domain of the NRPS module FscI in fuscachelin biosynthesis likely catalyzes amide bond formation at both the a- and -amines of a PCP-bound L-hOrn intermediate. Other domains displaying similar activity include the condensation domain of MxcG, the third C domain of CchH, and the second C domain of VibF from the biosynthetic pathways for myxochelin, coelichelin, and vibriobactin, respectively. 

In a limited number of NRPSs, the final module terminates in a specialized C domain that catalyzes chain release through amide bond formation. Modules of this type are found in the synthetases involved in the biosynthesis of enniatin, vibriobactin, cyclosporin/ HC-toxin/ and PF1032A. Unlike TE termination, this method of chain release does not utilize an acyl-ester intermediate. Most likely, the chain termination precursor is presented to the C domain as an aminoacyl-5-PCP substrate. Most of these specialized C domains... 

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.

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.

Nucleophilic attack by the amino group of the neighbouring aminoacyl thioester is catalysed by the C domain, and this results in amide (peptide) bond formation. Enzyme-controlled biosynthesis in this manner is a feature of many microbial peptides, especially those containing unusual amino acids not encoded by DNA and where post-translational modification (see Section 13.1) is unlikely. 

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