Solution structure of the pro-hormone convertase 1 pro-domain from Mus musculus

The solution structure of the mouse pro-hormone convertase (PC) 1 pro-domain was determined using heteronuclear NMR spectroscopy and is the first structure to be obtained for any of the domains in the convertase family. The ensemble of NMR-derived structures shows a well-ordered core consisting of a four-stranded antiparallel beta-sheet with two alpha-helices packed against one side of this sheet. Sequence homology suggests that the other eukaryotic PC pro-domains will have the same overall fold and most of the residues forming the hydrophobic core of PC1 are highly conserved within the PC family. However, some of the core residues are predicted by homology to be replaced by polar amino acid residues in other PC pro-domains and this may help to explain their marginal stability. Interestingly, the folding topology observed here is also seen for the pro-domain of bacterial subtilisin despite little or no sequence homology. Both the prokaryotic and eukaryotic structures have hydrophobic residues clustered on the solvent-accessible surface of their beta-sheets although the individual residue types differ. In the bacterial case this region is buried at the binding interface with the catalytic domain and, in the eukaryotic PC family, these surface residues are conserved. We therefore propose that the hydrophobic patch in the PC1 pro-domain is involved in the binding interface with its cognate catalytic domain in a similar manner to that seen for the bacterial system. The PC1 pro-domain structure also reveals potential mechanisms for the acid-induced dissociation of the complex between pro- and catalytic domains.     

The Functional Maturation of A Disintegrin and Metalloproteinase (ADAM) 9, 10, and 17 Requires Processing at a Newly Identified Proprotein Convertase (PC) Cleavage Site

Proenzyme maturation is a general mechanism to control the activation of enzymes. Catalytically active members of the A Disintegrin And Metalloprotease (ADAM) family of membrane-anchored metalloproteases are synthesized as proenzymes, in which the latency is maintained by their autoinhibitory pro-domains. A proteolytic processing then transforms the proenzyme into a catalytically active form. The removal of the pro-domain of ADAMs is currently thought to depend on processing at a canonical consensus site for the proprotein convertase Furin (RXXR) between the pro- and the catalytic domain. Here, we demonstrate that this previously described canonical site is a secondary cleavage site to a prerequisite cleavage in a newly characterized upstream PC site embedded within the pro-domain sequence. The novel upstream regulatory site is important for the maturation of several ADAM proenzymes. Mutations in the upstream regulatory site of ADAM17, ADAM10, and ADAM9 do not prevent pro-domain processing between the pro- and metalloprotease domain, but nevertheless, cause significantly reduced catalytic activity. Thus, our results have uncovered a novel functionally relevant PC processing site in the N-terminal part of the pro-domain that is important for the activation of these ADAMs. These results suggest that the novel PC site is part of a general mechanism underlying proenzyme maturation of ADAMs that is independent of processing at the previously identified canonical Furin cleavage site.     

The pro-domain of the zebrafish Nodal-related protein Cyclops regulates its signaling activities

Nodal proteins are secreted signaling factors of the transforming growth factor beta (TGFbeta) family with essential roles in embryonic development in vertebrates. Mutations affecting the Nodal factors have severe consequences in mammals and fish. Furthermore, increased Nodal levels have been associated with melanoma tumor progression. Like other TGFbeta-related proteins, Nodal factors consist of a pro-domain and a mature domain. The pro-domain of mouse Nodal protein stabilizes its precursor. However, the mechanisms by which the pro-domains exert their activities are unknown. Here, we characterize the zebrafish Nodal-related factor Cyclops (Cyc) and find unexpected functions for the pro-domain in regulating Cyc activity. We identified a lysosome-targeting region in the Cyc pro-domain that destabilizes the precursor and restricts Cyc activity, revealing the molecular basis for the short-range signaling activities of Cyc. We show that both the pro- and mature-domains of Cyc regulate its stability. We also characterize a mutation in the pro-domain of human NODAL (hNODAL) that underlies congenital heterotaxia. Heterologous expression of mutant hNODAL increases expression of Nodal-response genes. Our studies reveal unexpected roles for the pro-domain of the Nodal factors and provide a possible mechanism for familial heterotaxia.     

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.     

Stabilizing the subtilisin BPN' pro-domain by phage display selection: How restrictive is the amino acid code for maximum protein stability?

We have devised a procedure using monovalent phage display to select for stable mutants in the pro-domain of the serine protease, subtilisin BPN'. In complex with subtilisin, the pro-domain assumes a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated, however, the pro-domain is 97% unfolded. These experiments use combinatorial mutagenesis to select for stabilizing amino acid combinations at a particular structural locus and determine how many combinations are close to the maximum protein stability. The selection for stability is based on the fact that the independent stability of the pro-domain is very low and that binding to subtilisin is thermodynamically linked to folding. Two libraries of mutant pro-domains were constructed and analyzed to determine how many combinations of amino acids at a particular structural locus result in the maximum stability. A library comprises all combinations of four amino acids at a structural locus. Previous studies using combinatorial genetics have shown that many different combinations of amino acids can be accommodated in a selected locus without destroying function. The present results indicate that the number of sequence combinations at a structural locus, which are close to the maximum stability, is small. The most striking example is a selection at an interior locus of the pro-domain. After two rounds of phagemid selection, one amino acid combination is found in 40% of sequenced mutants. The most frequently selected mutant has a deltaG(unfolding) = 4 kcal/mol at 25 degrees C, an increase of 6 kcal/mol relative to the naturally occurring sequence. Some implications of these results on the amount of sequence information needed to specify a unique tertiary fold are discussed. Apart from possible implications on the folding code, the phage display selection described here should be useful in optimizing the stability of other proteins, which can be displayed on the phage surface.     

Epitope mapping of antigenic MUC1 peptides to breast cancer antibody fragment B27.29: a heteronuclear NMR study

MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant "reverse templates" of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], (1)H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including (15)N and (13)C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)(2). (15)N and (13)C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The (13)C(alpha) T(1) values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the (15)N- and (13)C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast cancer vaccine design.     

The NMR structure of 31mer RNA domain of Escherichia coli RNase P RNA using its non-uniformly deuterium labelled counterpart [the 'NMR-window' concept].

The NMR structure of a 31mer RNA constituting a functionally important domain of the catalytic RNase P RNA from Escherichia coli is reported. Severe spectral overlaps of the proton resonances in the natural 31mer RNA (1) were successfully tackled by unique spectral simplifications found in the partially-deuterated 31 mer RNA analogue (2) incorporating deuterated cytidines [C5 (>95 atom % 2H), C2' (>97 atom % 2H), C3' (>97 atom % 2H), C4' (>65 atom % 2H) and C5' (>97 atom % 2H)] [for the 'NMR-window' concept see: Földesi,A. et al. (1992) Tetrahedron, 48, 9033; Foldesi,A. et al. (1993) J. Biochem. Biophys. Methods, 26, 1; Yamakage,S.-I. et al. (1993) Nucleic Acids Res., 21, 5005; Agback,P. et al. (1994) Nucleic Acids Res., 22, 1404; Földesi,A. et al. (1995) Tetrahedron, 51, 10065; Földesi,A. et al. (1996) Nucleic Acids Res., 24, 1187-1194]. 175 resonances have been assigned out of total of 235 non-exchangeable proton resonances in (1) in an unprecedented manner in the absence of 13C and 15N labelling. 41 out of 175 assigned resonances could be accomplished with the help of the deuterated analogue (2). The two stems in 31mer RNA adopt an A-type RNA conformation and the base-stacking continues from stem I into the beginning of the loop I. Long distance cross-strand NOEs showed a structured conformation at the junction between stem I and loop I. The loop I-stem II junction is less ordered and shows structural perturbation at and around the G11 -C22 base pair.     

Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity

In eukaryotes, many secreted proteins and peptide hormones are excised from larger precursors by calcium-dependent serine proteinases, the proprotein/prohormone convertases (PCs). These PCs cleave their protein substrates very specifically following multiple basic residues. The seven mammalian PCs and their yeast orthologue kexin are multi-domain proteinases consisting of a subtilisin-related catalytic domain, a conserved P-domain and a variable, often cysteine-rich domain, which in some PCs is followed by an additional C-terminal trans-membrane domain and a short cytoplasmic domain. The recently published crystal structures of the soluble mouse furin and yeast kexin ectodomains have revealed the relative arrangement of catalytic and P domains, the exact domain fold and the detailed architecture of the substrate binding clefts. Based on these experimental structures, we now have modelled the structures of the other human/mouse PCs. According to topology and to structure-based sequence comparisons, these other PCs closely resemble furin, with PC4, PACE4 and PC5/6 being more similar, and PC1/3, PC2 and PC7 being less similar to furin. Except for PC1 and PC2, this order of similarity is valid for the catalytic as well as for the P domains, and is almost reversed using kexin as a reference molecule. A similar order results from the number and clustering of negative charges lining the non-prime subsites, explaining the gradually decreasing requirement for basic residues N-terminal to substrate cleavage sites. The preference of the different PCs for distinct substrates seems to be governed by overall charge compensation and matching of the detailed charge distribution pattern.     

High-resolution NMR study of a synthetic DNA-RNA hybrid dodecamer containing the consensus pribnow promoter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG)

The nonsymmetrical double-helical hybrid dodecamer d(CGTTATAATGCG).r(CGCAUUAUAACG) was synthesized with solid-phase phosphoramidite methods and studied by high-resolution 2D NMR. The imino protons were assigned by one-dimensional nuclear Overhauser methods. All the base protons and H1', H2', H2", H3', and H4' sugar protons of the DNA strand and the base protons, H1', H2', and most of the H3'-H4' protons of the RNA strand were assigned by 2D NMR techniques. The well-resolved spectra allowed a qualitative analysis of relative proton-proton distances in both strands of the dodecamer. The chemical shifts of the hybrid duplex were compared to those of the pure DNA double helix with the same sequence (Wemmer et al., 1984). The intrastrand and cross-strand NOEs from adenine H2 to H1' resonances of neighboring base pairs exhibited characteristic patterns that were very useful for checking the spectral assignments, and their highly nonsymmetric nature reveals that the conformations of the two strands are quite different. Detailed analysis of the NOESY and COSY spectra, as well as the chemical shift data, indicate that the RNA strand assumes a normal A-type conformation (C3'-endo) whereas the DNA strand is in the general S domain but not exactly in the normal C2'-endo conformation. The overall structure of this RNA-DNA duplex is different from that reported for hybrid duplexes in solution by other groups (Reid et al., 1983a; Gupta et al., 1985) and is closer to the C3'-endo-C2'-endo hybrid found in poly(dA).poly(dT) and poly(rU).poly(dA) in the fiber state (Arnott et al., 1983, 1986).     

Two-dimensional nMR study of bleomycin and its zinc(II) complex: reassignment of 13C resonances.

Two-dimensional NMR experiments--one bond 1H-13C correlation spectroscopy and heteronuclear multiple bond correlation spectroscopy, both performed in the reverse detection mode--have been employed to unambiguously assign all of the 13C resonances of the antibiotic bleomycin and its zinc(II) complex. Previous 1H resonance assignments of bleomycin (Chen et al. (1977) Biochemistry 16, 2731-2738) were confirmed on the basis of homonuclear Hartmann-Hahn and homonuclear COSY experiments. The 13C assignments differ substantially from those previously obtained by other investigators (Naganawa et al., (1977) J. Antibiot. 30, 388-396; Dabrowiak et al., (1978) Biochemistry 17, 4090-4096) but are in agreement with those reported by Akkerman et al. (1988) (Magn. Reson. Chem. 26, 793-802). The more recent study employed similar two-dimensional correlation experiments (performed in the direct detection mode) in conjunction with attached proton tests. Their study often required model compound data to identify carbonyls adjacent to aliphatic moieties. Previous 13C NMR studies of the structure, pH titration, and molecular dynamics of bleomycin and its zinc complex have been reinterpreted in terms of the revised assignments.     

C-terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retroviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 [Zn(HIV1-F2)]. Unlike results obtained for the first retroviral-type zinc finger peptide, Zn(HIV1-F1), [Summers et al. (1990) Biochemistry 29, 329], broad signals indicative of conformational lability were observed in the 1H NMR spectrum of Zn-(HIV1-F2) at 25 degrees C. The NMR signals narrowed upon cooling to -2 degrees C, enabling complete 1H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were used to generate 30 distance geometry (DG) structures with penalties (penalty = sum of the squared differences between interatomic distances defined in the restraints file and in the DG structures) in the range 0.02-0.03 A2. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. Superposition of the backbone atoms (C, C alpha, N) for residues C(1)-C(14) gave pairwise RMSD values in the range 0.16-0.75 A. The folding of Zn(HIV1-F2) is very similar to that observed for Zn(HIV1-F1). Small differences observed between the two finger domains are localized to residues between His(9) and Cys(14), with residues M(11)-C(14) forming a 3(10) helical corner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical shift assignments of Ribosomal protein S1 from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

RpsA, also known as ribosomal protein S1, is an essential protein required for translation initiation of mRNAs when their Shine-Dalgarno sequence is degenerated (Sorensen et al. 1998). In addition, RpsA of Mycobacterium tuberculosis (M. tb) is involved in trans-translation, which is an effective system mediated by tmRNA-SmpB to release stalled ribosomes from mRNA in the presence of rare codons (Keiler 2008). Shi et al. found that POA binds to RpsA of Mtb and disrupts the formation of RpsA–tmRNA complex (Shi et al. 2011) and mutations at the C-terminus of RpsA confer PZA resistance. The previous work reported the pyrazinoic acid-binding domain of RpsA (Yang et al. Mol Microbiol 95:791–803, 2015). However, the HSQC spectra of the isolated S1 domain does not overlap with that of MtRpsA280-438, suggesting that substantial interactions occur between the flexible C-terminus and the S1 domain in MtRpsA .To further study the PZA resistance and how substantial interactions influence/affect protein structure, using heteronuclear NMR spectroscopy, we have completed backbone and side-chain ¹H, ¹⁵N, ¹³C chemical shift assignments of MtRpsA280-438 which contains S1 domain and the flexible C-terminus. These NMR resonance assignments provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.     

Two-Dimensional NMR Study of Bleomycin and Its Zinc(II) Complex: Reassignment of 13C Resonances

Abstract

Two-dimensional NMR experiments-one bond ¹H- ¹³C correlation spectroscopy and hetero- nuclear multiple bond correlation spectroscopy, both performed in the reverse detection mode-have been employed to unambiguously assign all of the ¹³C resonances of the antibiotic bleomycin and its zinc(II) complex. Previous ¹H resonance assignments of bleomycin (Chen et al. (1977) Biochemistry 16, 2731–2738) were confirmed on the basis of homonuclear Hartmann-Hahn and homonuclear COSY experiments. The ¹³C assignments differ substantially from those previously obtained by other investigators (Naganawa et al., (1977) J. Antibiot. 30, 388–396; Dabrowiak et al., (1978) Biochemistry 17, 4090–4096) but are in agreement with those reported by Akkerman et al.(1988) (Magn. Reson. Chem. 26, 793–802). The more recent study employed similar two-dimensional correlation experiments (performed in the direct detection mode) in conjunction with attached proton tests. Their study often required model compound data to identify carbonyls adjacent to aliphatic moieties. Previous ¹³C NMR studies of the structure, pH titration, and molecular dynamics of bleomycin and its zinc complex have been reinterpreted in terms of the revised assignments.     

Structure of the Oct-3 POU-homeodomain in solution,as determined by triple resonance heteronuclear multidimensional NMR spectroscopy.

The POU-homeodomain (POUH) forms the bipartite DNA-binding POU domain in association with the POU-specific domain. The 1H, 15N, and 13C magnetic resonances of the 67-amino acid long POUH of mouse Oct-3 have almost completely been assigned, mainly through the combined use of three-dimensional triple resonance NMR methods. Based on the distance and dihedral angle constraints derived from the NMR data, the solution structure of the POUH domain has been calculated by the ab initio simulated annealing method. The average RMS deviation for all backbone heavy atoms of the 20 best calculated structures for residues 9-53 of the total 67 amino acid residues is 0.44 A. The POUH domain consists of three alpha-helices (helix-I, 10-20; helix-II, 28-38; and helix-III, 42-53), and helices-II and -III form a helix-turn-helix motif. In comparison with other classical homeodomains, the folding of the three helices is quite similar. However, the length of helix-III is fairly short. In the complex of the Oct-1 POU domain with an octamer site (Klemm JD, et al., 1994, Cell 77:21-32), the corresponding region is involved in helix-III. The structural difference between these two cases will be discussed.     

Multi-component analysis of marine lipids in fish gonads with emphasis on phospholipids using high resolution NMR spectroscopy

High resolution NMR has been applied for assessment of lipid classes and acyl stereospecific positions of fatty acids in marine phospholipids and triacylglycerols. 1D and 2D NMR techniques in combination with recording of a number of reference standards have been used to interpret the (1)H and (13)C NMR spectra of fish gonads. (13)C NMR spectra gave information regarding the polyunsaturated fatty acids (PUFAs) in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The carbonyl resonances showed that n-3 PUFAs primarily were esterified in the sn-2 position of PC and PE. The glycerol resonances showed that the PC/PE ratio was higher in roe than in milt and that roe comprised more triacylglycerols than milt. Thin layer chromatography showed that milt contained 2.4 times more cholesterol than roe, which was also found by integrating the (1)H NMR spectra. Concentration (mol%) of n-3 fatty acids were calculated from the (1)H NMR data and showed 44.8 and 36.3% in roe and milt, respectively.     

Structural similarities between MutT and the C-terminal domain of MutY

One of the functions of MutY from Escherchia coli is removal of adenine mispaired with 7,8-dihydro-8-oxoguanine (8-oxoG), a common lesion in oxidatively damaged DNA. MutY is composed of two domains: the larger N-terminal domain (p26) contains the catalytic properties of the enzyme while the C-terminal domain (p13) affects substrate recognition and enzyme turnover. On the basis of sequence analyses, it has been recently suggested that the C-terminal domain is distantly related to MutT, a dNTPase which hydrolyzes 8-oxo-dGTP [Noll et al. (1999) Biochemistry 38, 6374-6379]. We have studied the solution structure of the C-terminal domain of MutY by NMR and find striking similarity with the reported solution structure of MutT. Despite low sequence identity between the two proteins, they have similar secondary structure and topology. The C-terminal domain of MutY is composed of two alpha-helices and five beta-strands. The NOESY data indicate that the protein has two beta-sheets. MutT is also a mixed alpha/beta protein with two helices and two beta-sheets composed of five strands. The secondary structure elements are similarly arranged in the two proteins.     

Phosphatidyl-Tris rather than N-acylphosphatidylserine is synthesized by Rhodopseudomonas sphaeroides grown in Tris-containing media

We have synthesized 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(N-oleoyl)serine (N-acyl-PS) and 1,2-dioleoyl-sn-glycero-3-phospho-Tris (phosphatidyl-Tris) and have characterized both phospholipids by their chemical and chromatographic properties, as well as by their IR, 13C NMR, and 1H NMR spectra. Comparison of these data with those reported for a phospholipid isolated from Rhodopseudomonas sphaeroides grown in Tris-supplemented media [Donohue et al. (1982) Biochemistry 21, 2765-2773] indicates that R. sphaeroides synthesizes phosphatidyl-Tris rather than N-acyl-PS.     

Escherichia coli ilvN interacts with the FAD binding domain of ilvB and activates the AHAS I enzyme

The unique multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been exploited to generate polypeptide fragments which, when cloned and expressed, reassemble in the presence of cofactors to yield a catalytically competent enzyme. Multidimensional multinuclear NMR methods have been employed for obtaining near complete sequence specific NMR assignments for backbone HN, 15N, 13Calpha and 13Cbeta atoms of the FAD binding domain of ilvB on samples that were isotopically enriched in 2H, 13C and 15N. Unambiguous assignments were obtained for 169 of 177 backbone Calpha atoms and 127 of 164 side chain Cbeta atoms. The secondary structure determined on the basis of observed 13Calpha secondary chemical shifts and sequential NOEs agrees well with the structure of this domain in the catalytic subunit of yeast AHAS. Binding of ilvN to the ilvBalpha and ilvBbeta domains was studied by both circular dichroism and isotope edited solution nuclear magnetic resonance methods. Changes in CD spectra indicate that ilvN interacts with ilvBalpha and ilvBbeta domains of the catalytic subunit and not with the ilvBgamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvBbeta and proximal to the intrasubunit ilvBalpha/ilvBbeta domain interface. The implication of this interaction on the role of the regulatory subunit on the activity of the holoenzyme is discussed.