A General Model for Preferential Hetero-oligomerization of LIN-2/7 Domains
2005; Elsevier BV; Volume: 280; Issue: 46 Linguagem: Inglês
10.1074/jbc.m506536200
ISSN1083-351X
AutoresKeiko Y. Petrosky, Horng D. Ou, Frank Löhr, Volker Dötsch, Wendell A. Lim,
Tópico(s)Cellular Mechanics and Interactions
ResumoLIN-2/7 (L27) domains are protein interaction modules that preferentially hetero-oligomerize, a property critical for their function in directing specific assembly of supramolecular signaling complexes at synapses and other polarized cell-cell junctions. We have solved the solution structure of the heterodimer composed of the L27 domains from LIN-2 and LIN-7. Comparison of this structure with other L27 domain structures has allowed us to formulate a general model for why most L27 domains form an obligate heterodimer complex. L27 domains can be divided in two types (A and B), with each heterodimer comprising an A/B pair. We have identified two keystone positions that play a central role in discrimination. The residues at these positions are energetically acceptable in the context of an A/B heterodimer, but would lead to packing defects or electrostatic repulsion in the context of A/A and B/B homodimers. As predicted by the model, mutations of keystone residues stabilize normally strongly disfavored homodimers. Thus, L27 domains are specifically optimized to avoid homodimeric interactions. LIN-2/7 (L27) domains are protein interaction modules that preferentially hetero-oligomerize, a property critical for their function in directing specific assembly of supramolecular signaling complexes at synapses and other polarized cell-cell junctions. We have solved the solution structure of the heterodimer composed of the L27 domains from LIN-2 and LIN-7. Comparison of this structure with other L27 domain structures has allowed us to formulate a general model for why most L27 domains form an obligate heterodimer complex. L27 domains can be divided in two types (A and B), with each heterodimer comprising an A/B pair. We have identified two keystone positions that play a central role in discrimination. The residues at these positions are energetically acceptable in the context of an A/B heterodimer, but would lead to packing defects or electrostatic repulsion in the context of A/A and B/B homodimers. As predicted by the model, mutations of keystone residues stabilize normally strongly disfavored homodimers. Thus, L27 domains are specifically optimized to avoid homodimeric interactions. Accurate transmission of cell signaling information often requires the proper co-assembly of partner proteins (1.Park S.H. Zarrinpar A. Lim W.A. Science. 2003; 299: 1061-1064Crossref PubMed Scopus (291) Google Scholar). In eukaryotes, many of these assembly interactions are mediated by modular protein-protein interaction domains. Because these modular domains often compose large families of domains, discrimination between related domains is critical; lack of specificity leads to promiscuous interactions and improper signaling (2.Zarrinpar A. Park S.H. Lim W.A. Nature. 2003; 426: 676-680Crossref PubMed Scopus (213) Google Scholar). Thus, it is important to understand the molecular basis by which protein interaction domains recognize their correct partner but simultaneously discriminate against closely related but incorrect partners. Modular interaction domains play a central role in organizing polarized sites of cell-cell communication. At such sites of polarization, signaling proteins are organized into specific assemblies by scaffold proteins. The postsynaptic density in neurons (3.Kennedy M.B. Trends Neurosci. 1997; 20: 264-268Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 4.Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar), tight junction in epithelial cells (5.Mitic L.L. Anderson J.M. Annu. Rev. Physiol. 1998; 60: 121-142Crossref PubMed Scopus (628) Google Scholar), and immune synapse (6.Rudd C.E. Cell. 1999; 96: 5-8Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) are examples of highly organized signaling assembly coordinated by such scaffold proteins. In neurons and epithelial cells, PDZ (PSD95/DLG-1/ZO-1) domain-containing scaffold molecules play a central role in directing the trafficking and assembly of receptors and downstream effectors (7.Harris B.Z. Lim W.A. J. Cell Sci. 2001; 114: 3219-3231Crossref PubMed Google Scholar). More recently, many of these PDZ domain-containing scaffold proteins have been found to interact with one another to form higher order supramolecular complexes (8.Dimitratos S.D. Woods D.F. Stathakis D.G. Bryant P.J. BioEssays. 1999; 21: 912-921Crossref PubMed Scopus (197) Google Scholar, 9.Rongo C. Cytokine Growth Factor Rev. 2001; 12: 349-359Crossref PubMed Scopus (13) Google Scholar). These higher order interactions are mediated primarily by a novel type of protein interaction domain referred to as the LIN-2/7 (L27) 2The abbreviations used are: L27, LIN-2/7; GdnHCl, guanidine hydrochloride; HSQC, heteronuclear single quantum correlation; TROSY, transverse relaxation optimized spectroscopy; TOCSY, total correlation spectroscopy. domain (10.Kamberov E. Makarova O. Roh M. Liu A. Karnak D. Straight S. Margolis B. J. Biol. Chem. 2000; 275: 11425-11431Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 11.Harris B.Z. Venkatasubrahmanyam S. Lim W.A. J. Biol. Chem. 2002; 277: 34902-34908Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 12.Lee S. Fan S. Makarova O. Straight S. Margolis B. Mol. Cell. Biol. 2002; 22: 1778-1791Crossref PubMed Scopus (123) Google Scholar, 13.Roh M.H. Makarova O. Liu C.J. Shin K. Lee S. Laurinec S. Goyal M. Wiggins R. Margolis B. J. Cell Biol. 2002; 157: 161-172Crossref PubMed Scopus (299) Google Scholar, 14.Karnak D. Lee S. Margolis B. J. Biol. Chem. 2002; 277: 46730-46735Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) (Fig. 1A). L27 domains have been found only within metazoan scaffold proteins (15.Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (3028) Google Scholar), and several examples are shown in Fig. 1B. To date, there are 34 known human proteins containing L27 domains (15.Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (3028) Google Scholar). Several of these scaffold proteins form specific heterotrimer complexes (16.Kaech S.M. Whitfield C.W. Kim S.K. Cell. 1998; 94: 761-771Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 17.Jo K. Derin R. Li M. Bredt D.S. J. Neurosci. 1999; 19: 4189-4199Crossref PubMed Google Scholar, 18.Butz S. Okamoto M. Sudhof T.C. Cell. 1998; 94: 773-782Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 19.Borg J.P. Straight S.W. Kaech S.M. de Taddeo-Borg M. Kroon D.E. Karnak D. Turner R.S. Kim S.K. Margolis B. J. Biol. Chem. 1998; 273: 31633-31636Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), the assembly of which is mediated directly by L27 domains. For example, in Caenorhabditis elegans, the epidermal growth factor receptor LET-23 is targeted to the basolateral surface by a complex of the proteins LIN-2, LIN-7, and LIN-10 (16.Kaech S.M. Whitfield C.W. Kim S.K. Cell. 1998; 94: 761-771Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Similar complexes are found in mammalian cells (17.Jo K. Derin R. Li M. Bredt D.S. J. Neurosci. 1999; 19: 4189-4199Crossref PubMed Google Scholar, 18.Butz S. Okamoto M. Sudhof T.C. Cell. 1998; 94: 773-782Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 19.Borg J.P. Straight S.W. Kaech S.M. de Taddeo-Borg M. Kroon D.E. Karnak D. Turner R.S. Kim S.K. Margolis B. J. Biol. Chem. 1998; 273: 31633-31636Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). L27 domain-containing complexes are organized at neural muscular junctions (20.Thomas U. Kim E. Kuhlendahl S. Koh Y.H. Gundelfinger E.D. Sheng M. Garner C.C. Budnik V. Neuron. 1997; 19: 787-799Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 21.Bachmann A. Timmer M. Sierralta J. Pietrini G. Gundelfinger E.D. Knust E. Thomas U. J. Cell Sci. 2004; 117: 1899-1909Crossref PubMed Scopus (61) Google Scholar), epithelial cell contacts (22.Hong Y. Stronach B. Perrimon N. Jan L.Y. Jan Y.N. Nature. 2001; 414: 634-638Crossref PubMed Scopus (205) Google Scholar, 23.Bachmann A. Schneider M. Theilenberg E. Grawe F. Knust E. Nature. 2001; 414: 638-643Crossref PubMed Scopus (237) Google Scholar, 24.Shin K. Straight S. Margolis B. J. Cell Biol. 2005; 168: 705-711Crossref PubMed Scopus (149) Google Scholar, 25.Straight S.W. Shin K. Fogg V.C. Fan S. Liu C.J. Roh M. Margolis B. Mol. Biol. Cell. 2004; 15: 1981-1990Crossref PubMed Scopus (140) Google Scholar), and immune synapses (26.Ralston K.J. Hird S.L. Zhang X. Scott J.L. Jin B. Thorne R.F. Berndt M.C. Boyd A.W. Burns G.F. J. Biol. Chem. 2004; 279: 33816-33828Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 27.Round J.L. Tomassian T. Zhang M. Patel V. Schoenberger S.P. Miceli M.C. J. Exp. Med. 2005; 201: 419-430Crossref PubMed Scopus (101) Google Scholar). Thus, L27 domains appear to play a central role in the higher order organization of multi-scaffold assemblies. Biochemical studies have revealed that known L27 domains are obligate hetero-oligomerization units (11.Harris B.Z. Venkatasubrahmanyam S. Lim W.A. J. Biol. Chem. 2002; 277: 34902-34908Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). L27 domains, which are ∼60 residues (28.Doerks T. Bork P. Kamberov E. Makarova O. Muecke S. Margolis B. Trends Biochem. Sci. 2000; 25: 317-318Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), are largely unfolded in isolation, but fold to form a helical heterodimer upon interaction with the proper heterotypic partner (11.Harris B.Z. Venkatasubrahmanyam S. Lim W.A. J. Biol. Chem. 2002; 277: 34902-34908Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In some cases, two heterodimers can further oligomerize to form a higher order tetramer (29.Feng W. Long J.F. Fan J.S. Suetake T. Zhang M. Nat. Struct. Mol. Biol. 2004; 11: 475-480Crossref PubMed Scopus (75) Google Scholar, 30.Li Y. Karnak D. Demeler B. Margolis B. Lavie A. EMBO J. 2004; 23: 2723-2733Crossref PubMed Scopus (38) Google Scholar, 31.Feng W. Long J.F. Zhang M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6861-6866Crossref PubMed Scopus (24) Google Scholar). However, in all cases, all assemblies are built from the fundamental unit of a heterodimer. This core dimer unit displays an exclusive preference for heterodimerization versus homodimerization: typically, when only one L27 domain molecule is present, there is no evidence for folding into a homomer structure (11.Harris B.Z. Venkatasubrahmanyam S. Lim W.A. J. Biol. Chem. 2002; 277: 34902-34908Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The obligate heteromeric assembly of L27 domains is thought to be fundamental to its basic function of directing the formation of specific heteromeric supramolecular assemblies. The structures of several L27 domain-containing complexes have been reported (29.Feng W. Long J.F. Fan J.S. Suetake T. Zhang M. Nat. Struct. Mol. Biol. 2004; 11: 475-480Crossref PubMed Scopus (75) Google Scholar, 30.Li Y. Karnak D. Demeler B. Margolis B. Lavie A. EMBO J. 2004; 23: 2723-2733Crossref PubMed Scopus (38) Google Scholar), and mutational analysis has indicated that hydrophobic residues are involved in shape-complementary interactions across the dimer interface (30.Li Y. Karnak D. Demeler B. Margolis B. Lavie A. EMBO J. 2004; 23: 2723-2733Crossref PubMed Scopus (38) Google Scholar). However, little is understood about how L27 domains bind tightly to their correct heterotypic partners while showing almost no self-association. Here, we report the solution structure of the heterodimer composed of the LIN-7 L27 domain and the C-terminal L27 domain from LIN-2 (referred to as LIN-2C). By comparing this and other L27 domain heterodimer structures, we have formulated a general model for why most L27 domains form obligate heterodimers. L27 domains can be divided in two types, referred to as A and B (29.Feng W. Long J.F. Fan J.S. Suetake T. Zhang M. Nat. Struct. Mol. Biol. 2004; 11: 475-480Crossref PubMed Scopus (75) Google Scholar), with each heterodimer comprising an A/B pair. We have identified two keystone positions that play a central role in discrimination between heterodimer and homodimer formation. The residues at these positions are energetically acceptable in the context of an A/B heterodimer, but would be destabilizing in the context of a hypothetical A/A or B/B homodimer, leading to packing defects or electrostatic repulsion. As predicted by the model, mutation of these keystone positions increases the stability of the normally strongly disfavored homodimers. Thus, L27 domains are specifically optimized by negative design: they are simultaneously optimized to maximize heterotypic complementarity and to avoid homotypic interactions. Cloning and Expression of Linked L27 Domains—DNA regions encoding L27 domains were amplified by PCR (11.Harris B.Z. Venkatasubrahmanyam S. Lim W.A. J. Biol. Chem. 2002; 277: 34902-34908Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The regions cloned comprised C. elegans LIN-7A amino acids 115-180 (referred to as LIN-7) and H. sapiens LIN-2 amino acids 394-460 (referred to as LIN-2C). The mixed species LIN-7/LIN-2C pair (C. elegans/Homo sapiens) was previously shown to be particularly stable (11.Harris B.Z. Venkatasubrahmanyam S. Lim W.A. J. Biol. Chem. 2002; 277: 34902-34908Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and best suited for structural analysis. L27 domains from LIN-7 and LIN-2C were expressed as a linked construct to ensure a 1:1 ratio and to improve solubility, a technique previously used to express the SAP97/LIN-2N L27 domain pair (29.Feng W. Long J.F. Fan J.S. Suetake T. Zhang M. Nat. Struct. Mol. Biol. 2004; 11: 475-480Crossref PubMed Scopus (75) Google Scholar). PCR primers where used to engineer a Gly-Ser linker between the two L27 domains. The coding fragments were ligated into the expression vector pBH4, which provided an N-terminal His6 tag for purification. All constructs were verified by sequencing on both strands. The LIN-7 L27 domain construct has been described previously (11.Harris B.Z. Venkatasubrahmanyam S. Lim W.A. J. Biol. Chem. 2002; 277: 34902-34908Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Keystone residues were mutated by QuikChange site-directed mutagenesis. Protein constructs were expressed in Escherichia coli strain BL21 pLysS (Invitrogen). Cultures were grown in standard Luria broth at 32 °C and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside at A600 nm ∼ 0.8. To obtain labeled protein constructs (15N, 13C, and 13C,15N; 70% deuterated) for NMR analysis, cultures were transferred to M9 minimal medium containing labeled 15NH4Cl and/or [13C6]glucose before induction. Unlabeled protein constructs were harvested by centrifugation after 3 h of growth, whereas labeled protein constructs were harvested after 5 h. Cell pellets were resuspended in ∼25 ml of lysis buffer (50 mm Tris-Cl (pH 7.5) and 400 mm NaCl) per original liter of culture and subjected to freeze-thaw treatment. Cell suspensions were lysed by sonication and cleared by centrifugation at 15,000 × g. Wild-type and mutant constructs were expressed at similar levels. The His6 tag-containing proteins were purified under native conditions by nickel-nitrilotriacetic acid affinity chromatography (Qiagen Inc.). For NMR analysis, purified protein was dialyzed in 20 mm HEPES (pH 8.0) and 0.5 mm tris(2-carboxyethyl)phosphine hydrochloride for constructs containing cysteines, concentrated, and stored at -80 °C until used. For CD experiments, protein was stored in 50 mm phosphate buffer (pH 7.5), 10 mm NaCl, and 1 mm tris(2-carboxyethyl)phosphine hydrochloride for constructs containing cysteines. NMR Structure Determination and Analysis—NMR experiments were carried out at 25 °C on Bruker DRX500, DMX600, and Avance 700 spectrometers equipped with conventional 1H{13C/15N} triple resonance probes and on Bruker DRX600, Avance800, and Avance900 spectrometers equipped with cryogenic 1H{13C/15N} probes. Samples contained 2 mm protein in 90% H2O and 10% D2O with 20 mm HEPES (pH 8.0), 5 mm tris(2-carboxyethyl)phosphine hydrochloride, 0.05% 2,2-dimethylsilapentane-5-sulfonic acid, and 0.05% sodium azide. Signal dispersion of the constructs was verified with 15N heteronuclear single quantum correlation spectroscopy (HSQC) spectra. Sequential backbone assignments were derived from transverse relaxation optimized spectroscopy (TROSY)-type three-dimensional heteronuclear correlation experiments, including HNCO, HN(CA)CO, HN(CO)CACB, and HNCACB (32.Salzmann M.W.G. Pervushin K. Senn H. Wuthrich K. J. Am. Chem. Soc. 1999; 121: 844-848Crossref Scopus (296) Google Scholar, 33.Deleted in proofGoogle Scholar). Side chains were assigned through a combination of total correlation spectroscopy (TOCSY)-[15N,1H]-TROSY, (H)CC(CO)NH and HCC(CO)NH spectra (32.Salzmann M.W.G. Pervushin K. Senn H. Wuthrich K. J. Am. Chem. Soc. 1999; 121: 844-848Crossref Scopus (296) Google Scholar). Distance constraints were derived from nuclear Overhauser effect correlation spectroscopy (NOESY)-[15N,1H]-TROSY, two types of NOESY-[13C,1H]-HSQC experiments optimized for CH3 groups and aliphatic CH/CH2 groups and a constant time NOESY-[13C,1H]-TROSY with mixing times ranging 70 to 120 ms (32.Salzmann M.W.G. Pervushin K. Senn H. Wuthrich K. J. Am. Chem. Soc. 1999; 121: 844-848Crossref Scopus (296) Google Scholar). Spectra were processed using XWIN-NMR (Bruker BioSpin Corp.) and analyzed using XEASY (34.Bartels C. Xia T. Billeter M. Güntert P. Wüthrich K. J. Biomol. NMR. 1995; 6: 1-10Crossref PubMed Scopus (1607) Google Scholar). Chemical shifts were referenced to 2,2-dimethylsilapentane-5-sulfonic acid. Backbone dihedral angle restraints were derived from N, HN, C-α, and C-β chemical shifts with the program TALOS (35.Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar). Structures were iteratively refined using the simulated annealing protocol of the torsion angle dynamics program DYANA (36.Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2558) Google Scholar). Nuclear Overhauser effect peak intensities were converted into upper distance bounds with the CALIBA function of DYANA. Of the final 100 structures calculated, the 10 conformers with the lowest target function values were selected for analysis. These structures were averaged, and the resulting structure was minimized in CNS (37.Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16978) Google Scholar) using conjugate gradient minimization. The resulting 10 best structures were assessed for overall quality using PROCHECK (see Fig. 2) (38.Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4469) Google Scholar). The coordinates have been deposited in the Protein Data Bank (code 1ZL8). The residues in the Protein Data Bank file (Leu5-Tyr53 from the L27 domain of LIN-7 and Ser6-Ala59 from the C-terminal domain of LIN-2) correspond to the numbering in Fig. 3. Note that residues not observed by NMR are not included in the Protein Data Bank file. Figures were generated using MolMol (39.Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6497) Google Scholar) and PyMOL (40.DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). Contact maps were generated using CNS.FIGURE 3Sequence divergence between A- and B-type L27 domains. A, L27 domain sequences cluster into A- and B-type domains by sequence homology; B, sequence alignment of A- and B-type domains. We have identified two differentiating "keystone" positions. At the "electrostatic" keystone position, A-type domains generally have a positively charged residue (+), whereas B-type domains have a negatively charged residue (-). A subclass of A-type domains (SAP97 family members) have a neutral residue (N), the implications of which are reviewed under "Discussion." At the "hydrophobic/polar" keystone position, A-type domains have a conserved hydrophobic residue (H), which is polar in the B-type domains (P).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Circular Dichroism—CD spectroscopy was performed on a Jasco 715 spectropolarimeter. All scans were performed in a 1-cm quartz cuvette in 50 mm sodium phosphate buffer (pH 7.5), 10 mm NaCl, and 1 mm tris(2-carboxyethyl)phosphine hydrochloride for constructs containing cysteines. The CD spectra shown are the averages of two experiments. Data for wavelength scans were collected at 1-nm intervals with 1-s averaging time/data point and a total of 10 repetitions/scan. Data for guanidine hydrochloride (GdnHCl) denaturation studies were collected at 222 nm with 0.1 m denaturant intervals and with 60-s averaging time/data point and 60-s equilibration time/data point. Identical results were obtained with the wild-type protein for longer equilibration times (data not shown). Both orientations of linked domains were found to behave identically in denaturant melts (data not shown). The midpoint of the unfolding transition is concentration-independent above 4 μm for the wild-type construct (data not shown). For all experiments, the total L27 domain concentration was 10 μm (10 μm monomers and 5 μm linked heterodimer). Structure of the LIN-7/LIN-2C L27 Domain Heterodimer—The structure of the LIN-7/LIN-2C heterodimer was solved by NMR as shown in Fig. 2. The two domains are expressed in a single covalently linked protein that yields improved expression and provides a 1:1 stoichiometry while not perturbing the structure of the complex (31.Feng W. Long J.F. Zhang M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6861-6866Crossref PubMed Scopus (24) Google Scholar). The unlinked LIN-7·LIN-2C complex was previously shown to exist primarily as a dimer (11.Harris B.Z. Venkatasubrahmanyam S. Lim W.A. J. Biol. Chem. 2002; 277: 34902-34908Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). When covalently linked, the apparent molecular mass of the complex doubles (data not shown), consistent with formation of a tetramer at NMR and CD concentrations. A similar tetramer structure has recently been reported for a LIN-7·LIN-2C complex from mouse (31.Feng W. Long J.F. Zhang M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6861-6866Crossref PubMed Scopus (24) Google Scholar). The tetramer interface is discussed elsewhere in great detail (29.Feng W. Long J.F. Fan J.S. Suetake T. Zhang M. Nat. Struct. Mol. Biol. 2004; 11: 475-480Crossref PubMed Scopus (75) Google Scholar, 31.Feng W. Long J.F. Zhang M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6861-6866Crossref PubMed Scopus (24) Google Scholar); here, we focused primarily on the interaction specificity within the core heterodimer unit. The structure of the core heterodimer is similar to those of the previously solved L27 domain heterodimers, those involving SAP97/LIN-2N (29.Feng W. Long J.F. Fan J.S. Suetake T. Zhang M. Nat. Struct. Mol. Biol. 2004; 11: 475-480Crossref PubMed Scopus (75) Google Scholar) and PATJ/PALS1N (30.Li Y. Karnak D. Demeler B. Margolis B. Lavie A. EMBO J. 2004; 23: 2723-2733Crossref PubMed Scopus (38) Google Scholar). (LIN-2 and PALS1 scaffold proteins each contain two L27 domains; the "N" indicates that these structures contain the N-terminal L27 domain.) Each individual monomer contains three α-helices, with the first two helices from each monomer packing together to form a four-helix bundle that forms the core of the dimer. The third helices from the two monomers pack against one another and the bottom of the helical bundle, capping the hydrophobic core presented by the bundle. The overall heterodimer structure buries a surface area of 2159 Å2, 69% of which is due to the packing interface of the central four-helix bundle. L27 Domains Fall into Two Distinct Sequence Types—When aligned, all known L27 domain sequences segregate into two distinct types, referred to as A and B (29.Feng W. Long J.F. Fan J.S. Suetake T. Zhang M. Nat. Struct. Mol. Biol. 2004; 11: 475-480Crossref PubMed Scopus (75) Google Scholar). The clustering of these two types is shown in a dendrogram and by sequence alignment in Fig. 3 (A and B). Strikingly, each pair of known interacting L27 domains involves the interaction of an A-type monomer with a B-type monomer (10.Kamberov E. Makarova O. Roh M. Liu A. Karnak D. Straight S. Margolis B. J. Biol. Chem. 2000; 275: 11425-11431Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 11.Harris B.Z. Venkatasubrahmanyam S. Lim W.A. J. Biol. Chem. 2002; 277: 34902-34908Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 12.Lee S. Fan S. Makarova O. Straight S. Margolis B. Mol. Cell. Biol. 2002; 22: 1778-1791Crossref PubMed Scopus (123) Google Scholar, 13.Roh M.H. Makarova O. Liu C.J. Shin K. Lee S. Laurinec S. Goyal M. Wiggins R. Margolis B. J. Cell Biol. 2002; 157: 161-172Crossref PubMed Scopus (299) Google Scholar, 14.Karnak D. Lee S. Margolis B. J. Biol. Chem. 2002; 277: 46730-46735Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Not only are most monomers unable to self-associate, but they are also unable to interact with distinct monomers from within the same type (11.Harris B.Z. Venkatasubrahmanyam S. Lim W.A. J. Biol. Chem. 2002; 277: 34902-34908Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 12.Lee S. Fan S. Makarova O. Straight S. Margolis B. Mol. Cell. Biol. 2002; 22: 1778-1791Crossref PubMed Scopus (123) Google Scholar, 14.Karnak D. Lee S. Margolis B. J. Biol. Chem. 2002; 277: 46730-46735Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In the case of the LIN-7/LIN-2C heterodimer studied here, LIN-7 is the A-type monomer, whereas LIN-2C is a B-type monomer. Conserved Structural Asymmetry in L27 Domain Dimers—A comparison of the three known L27 domain dimer structures is shown in Fig. 4A. These structures align well (LIN-7/LIN-2C versus PATJ/PALS1N root mean square deviation (C-α) = 2.17 Å and LIN-7/LIN-2C versus SAP97/LIN-2N root mean square deviation (C-α) = 3.45 Å) only when the same monomer subtypes are aligned (Fig. 4B). The other chains in the tetramer structures are similarly aligned. Careful examination revealed that there are distinct asymmetries between the A- and B-type monomer structures that are conserved across all known structures (31.Feng W. Long J.F. Zhang M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6861-6866Crossref PubMed Scopus (24) Google Scholar). Several key asymmetric features are highlighted in the contact maps shown in Fig. 4B. For the individual monomer units, in the B-type monomer, helix B1 and B3 come into close contact, whereas in the A-type monomer, helix A1 and A3 do not. In the dimer structure, helix B3 comes within 6 Å of helix A2, whereas helix A3 does not come closer than 10 Å to helix B2. These asymmetries can be understood by examining the overall dimer structure (Fig. 4A). Although helices A3 and B3 function together to cap the bottom end of the central four-helix bundle, helix B3 packs directly against the base of the bundle. In contrast, helix A3 primarily packs against helix B3. Keystone Positions: Potential Determinants of A- and B-type Monomer Structural Identity—The well conserved structural asymmetry between A- and B-type monomers in three different L27 domain dimer structures suggests that there is a fundamental difference in monomer sequence that results in these clear structural differences. Thus, we carefully examined the sequences of A and B subtypes for potential keystone positions that are distinct in each type. Two strong candidate positions emerged. First, the third residue in helix 2 has distinct electrostatic properties; in A-type monomers, this residue is either positively charged or neutral, whereas in B-type monomers, it is almost always negatively charged. Additionally, the second residue in helix 3 has distinct hydrophobic versus polar character; in A-type monomers, this position is always a large hydrophobic residue, whereas in B-type monomers, it is a polar residue. These potential keystone positions are highlighted in the alignment shown in Fig. 3B. In the following two sections, we examine how these two keystone positions might contribute to preferential heterodimerization. The Hydrophobic/Polar Keystone Position—The second residue in helix 3 is generally a large hydrophobic residue (Phe, Leu, or Met) in A-type L27 domains, whereas it is almost always a polar residue (His, Asp, or Ser) in B-type L27 domains (Fig. 3). Examination of L27 domain dimer structures revealed that this position is at the center of the conserved structural asymmetry. Helix A3 adopts a different orientation compared with helix B3 with respect to the main four-helix bundle. Because of this different orientation, as shown in Fig. 5A, the second residue in helix A3 (e.g. Phe41 in LIN-7) points into the core of the protein, whereas the second residue in helix B3 (e.g. His39 in LIN-2C) points toward the solvent. Thus, a possible model is that, to satisfy packing and hydrophobicity requirements, this position must be a hydrophobic residue in the A-type monomer, whereas t
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