Interplay between hevin, SPARC, and MDGAs: Modulators of neurexin-neuroligin transsynaptic bridges

Abstract

•An elaborate protein network regulates neurexin-neuroligin transsynaptic bridges•The hevin FS domain binds both neurexin and neuroligin via a direct interaction•Antagonist SPARC also binds neurexin/neuroligin•Hevin and MDGAs, which exert opposite actions, compete for binding to neuroligin Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges. Here, we reveal the molecular underpinnings of a regulatory network formed by this trio of proteins. The hevin FS-EC structure differs from SPARC, in that the EC domain appears rearranged around a conserved core. The FS domain is structurally conserved and it houses nanomolar affinity binding sites for neurexin and neuroligin. SPARC also binds neurexin and neuroligin, competing with hevin, so its antagonist action is rooted in its shortened N-terminal region. Strikingly, the hevin FS domain competes with MDGA for an overlapping binding site on neuroligin, while the hevin EC domain binds the extracellular matrix protein collagen (like SPARC), so that this trio of proteins can regulate neurexin-neuroligin transsynaptic bridges and also extracellular matrix interactions, impacting synapse formation and ultimately neural circuits. Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges. Here, we reveal the molecular underpinnings of a regulatory network formed by this trio of proteins. The hevin FS-EC structure differs from SPARC, in that the EC domain appears rearranged around a conserved core. The FS domain is structurally conserved and it houses nanomolar affinity binding sites for neurexin and neuroligin. SPARC also binds neurexin and neuroligin, competing with hevin, so its antagonist action is rooted in its shortened N-terminal region. Strikingly, the hevin FS domain competes with MDGA for an overlapping binding site on neuroligin, while the hevin EC domain binds the extracellular matrix protein collagen (like SPARC), so that this trio of proteins can regulate neurexin-neuroligin transsynaptic bridges and also extracellular matrix interactions, impacting synapse formation and ultimately neural circuits. Hevin (high endothelial venule protein), also known as SPARCL1 (SPARC-like 1, SC1, secreted protein acidic and rich in cysteine-like 1), is a secreted matricellular protein that promotes the formation and maintenance of neural circuits in mammalian brain by altering synaptic connections between neurons as well as impacting the position of neurons as they migrate (Allen and Eroglu, 2017Allen N.J. Eroglu C. Cell biology of astrocyte-synapse interactions.Neuron. 2017; 96: 697-708Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar; Ferrer-Ferrer and Dityatev, 2018Ferrer-Ferrer M. Dityatev A. Shaping synapses by the neural extracellular matrix.Front. Neuroanat. 2018; 12: 40Crossref PubMed Scopus (69) Google Scholar; Jones and Bouvier, 2014Jones E.V. Bouvier D.S. Astrocyte-secreted matricellular proteins in CNS remodelling during development and disease.Neural Plast. 2014; 2014: 321209Crossref PubMed Scopus (106) Google Scholar; Yuzaki, 2018Yuzaki M. Two classes of secreted synaptic organizers in the central nervous system.Annu. Rev. Physiol. 2018; 80: 243-262Crossref PubMed Scopus (50) Google Scholar). Hevin is composed of an N-terminal thread-like, flexible acidic region and a C-terminal globular region containing a follistatin-like (FS) domain and an extracellular calcium-binding (EC) domain (Hambrock et al., 2003Hambrock H.O. Nitsche D.P. Hansen U. Bruckner P. Paulsson M. Maurer P. Hartmann U. SC1/hevin. An extracellular calcium-modulated protein that binds collagen I.J. Biol. Chem. 2003; 278: 11351-11358Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The FS-EC tandem has ∼61% sequence identity to the matricellular protein SPARC (secreted protein acidic and rich in cysteine), a much smaller protein with a very short N-terminal region that is not conserved in hevin (Bradshaw, 2012Bradshaw A.D. Diverse biological functions of the SPARC family of proteins.Int. J. Biochem. Cell Biol. 2012; 44: 480-488Crossref PubMed Scopus (158) Google Scholar; Girard and Springer, 1995Girard J.P. Springer T.A. Cloning from purified high endothelial venule cells of hevin, a close relative of the antiadhesive extracellular matrix protein SPARC.Immunity. 1995; 2: 113-123Abstract Full Text PDF PubMed Scopus (143) Google Scholar; Murphy-Ullrich and Sage, 2014Murphy-Ullrich J.E. Sage E.H. Revisiting the matricellular concept.Matrix Biol. 2014; 37: 1-14Crossref PubMed Scopus (239) Google Scholar). Proteins that induce and/or maintain synapses are an important focal point of study because they are increasingly linked to neuropsychiatric disorders (Südhof, 2017Südhof T.C. Synaptic neurexin complexes: a molecular code for the logic of neural circuits.Cell. 2017; 171: 745-769Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), but for many, including hevin, relatively little is known about their molecular mechanisms. Hevin and SPARC have both similar as well as opposing biological functions in the brain. Hevin stimulates excitatory synapse formation (Gan and Südhof, 2019Gan K.J. Südhof T.C. Specific factors in blood from young but not old mice directly promote synapse formation and NMDA-receptor recruitment.Proc. Natl. Acad. Sci. U S A. 2019; 116: 12524-12533Crossref PubMed Scopus (39) Google Scholar; Gan and Südhof, 2020Gan K.J. Südhof T.C. SPARCL1 promotes excitatory but not inhibitory synapse formation and function independent of neurexins and neuroligins.J. Neurosci. 2020; 40: 8088-8102Crossref PubMed Scopus (16) Google Scholar; Kucukdereli et al., 2011Kucukdereli H. Allen N.J. Lee A.T. Feng A. Ozlu M.I. Conatser L.M. Chakraborty C. Workman G. Weaver M. Sage E.H. et al.Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.Proc. Natl. Acad. Sci. U S A. 2011; 108: E440-E449Crossref PubMed Scopus (329) Google Scholar). Its action is important to generate refined neural circuits; for instance, hevin secreted by astrocytes promotes and stabilizes thalamocortical excitatory synapses in the developing mouse visual cortex, strengthening the connections between the two brain regions, at the cost of the intracortical excitatory connections, which are eliminated (Kucukdereli et al., 2011Kucukdereli H. Allen N.J. Lee A.T. Feng A. Ozlu M.I. Conatser L.M. Chakraborty C. Workman G. Weaver M. Sage E.H. et al.Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.Proc. Natl. Acad. Sci. U S A. 2011; 108: E440-E449Crossref PubMed Scopus (329) Google Scholar; Risher et al., 2014Risher W.C. Patel S. Kim I.H. Uezu A. Bhagat S. Wilton D.K. Pilaz L.-J. Singh Alvarado J. Calhan O.Y. Silver D.L. et al.Astrocytes refine cortical connectivity at dendritic spines.Elife. 2014; 3: e04047Crossref Scopus (87) Google Scholar; Singh et al., 2016Singh S.K. Stogsdill J.A. Pulimood N.S. Dingsdale H. Kim Y.H. Pilaz L.-J. Kim I.H. Manhaes A.C. Rodrigues W.S. Pamukcu A. et al.Astrocytes assemble thalamocortical synapses by bridging NRX1α and NL1 via hevin.Cell. 2016; 164: 183-196Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). SPARC also affects synapse formation, albeit differently than hevin (Bradshaw, 2012Bradshaw A.D. Diverse biological functions of the SPARC family of proteins.Int. J. Biochem. Cell Biol. 2012; 44: 480-488Crossref PubMed Scopus (158) Google Scholar; Murphy-Ullrich and Sage, 2014Murphy-Ullrich J.E. Sage E.H. Revisiting the matricellular concept.Matrix Biol. 2014; 37: 1-14Crossref PubMed Scopus (239) Google Scholar). SPARC lacks the ability to induce synaptogenesis and it blocks hevin action (Kucukdereli et al., 2011Kucukdereli H. Allen N.J. Lee A.T. Feng A. Ozlu M.I. Conatser L.M. Chakraborty C. Workman G. Weaver M. Sage E.H. et al.Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.Proc. Natl. Acad. Sci. U S A. 2011; 108: E440-E449Crossref PubMed Scopus (329) Google Scholar). Interestingly, a fragment of hevin, SLF (SPARC-like fragment), that encompasses the FS and EC domains just like SPARC, is also not synaptogenic and likewise blocks hevin-induced synapse formation (Kucukdereli et al., 2011Kucukdereli H. Allen N.J. Lee A.T. Feng A. Ozlu M.I. Conatser L.M. Chakraborty C. Workman G. Weaver M. Sage E.H. et al.Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.Proc. Natl. Acad. Sci. U S A. 2011; 108: E440-E449Crossref PubMed Scopus (329) Google Scholar). SPARC triggers a cell-autonomous program of synapse elimination in cholinergic neurons (López-Murcia et al., 2015López-Murcia F.J. Terni B. Llobet A. SPARC triggers a cell-autonomous program of synapse elimination.Proc. Natl. Acad. Sci. U S A. 2015; 112: 13366-13371Crossref PubMed Scopus (31) Google Scholar), although in single-cell cholinergic neuron microcultures it increases the formation of autapses (i.e., synapses from one neuron onto itself) (Albrecht et al., 2012Albrecht D. López-Murcia F.J. Pérez-González A.P. Lichtner G. Solsona C. Llobet A. SPARC prevents maturation of cholinergic presynaptic terminals.Mol. Cell. Neurosci. 2012; 49: 364-374Crossref PubMed Scopus (26) Google Scholar). SPARC also regulates the efficiency of synaptic communication in developing hippocampus by controlling the number of AMPA-receptor subunits at synapses via a β3-integrin-mediated mechanism, while hevin does not (Jones et al., 2011Jones E.V. Bernardinelli Y. Tse Y.C. Chierzi S. Wong T.P. Murai K.K. Astrocytes control glutamate receptor levels at developing synapses through SPARC-beta-integrin interactions.J. Neurosci. 2011; 31: 4154-4165Crossref PubMed Scopus (85) Google Scholar). However, both hevin and SPARC alter neurite outgrowth (thus influencing neuron morphology), as well as neuronal migration and connectivities during postnatal development (López-Murcia et al., 2015López-Murcia F.J. Terni B. Llobet A. SPARC triggers a cell-autonomous program of synapse elimination.Proc. Natl. Acad. Sci. U S A. 2015; 112: 13366-13371Crossref PubMed Scopus (31) Google Scholar; Vincent et al., 2008Vincent A.J. Lau P.W. Roskams A.J. SPARC is expressed by macroglia and microglia in the developing and mature nervous system.Dev. Dyn. 2008; 237: 1449-1462Crossref PubMed Scopus (53) Google Scholar), perhaps via interactions with the extracellular matrix. SPARC has a well-described role in extracellular matrix assembly through its ability to bind collagen, which has been elucidated by structural studies. Whether hevin binds collagen as well is unclear (Bradshaw, 2012Bradshaw A.D. Diverse biological functions of the SPARC family of proteins.Int. J. Biochem. Cell Biol. 2012; 44: 480-488Crossref PubMed Scopus (158) Google Scholar; Hohenester et al., 2008Hohenester E. Sasaki T. Giudici C. Farndale R.W. Bächinger H.P. Structural basis of sequence-specific collagen recognition by SPARC.Proc. Natl. Acad. Sci. U S A. 2008; 105: 18273-18277Crossref PubMed Scopus (104) Google Scholar; Murphy-Ullrich and Sage, 2014Murphy-Ullrich J.E. Sage E.H. Revisiting the matricellular concept.Matrix Biol. 2014; 37: 1-14Crossref PubMed Scopus (239) Google Scholar). In the brain, hevin and SPARC show characteristic expression patterns that are temporally and spatially regulated, placing these molecules in highly strategic positions to modulate neural circuit formation and maintenance during development and beyond. Throughout postnatal development, hevin is abundantly expressed in astrocytes and in subsets of projection neurons, escalating to high levels during a peak period of synaptic remodeling (Lively and Brown, 2008aLively S. Brown I.R. Localization of the extracellular matrix protein SC1 coincides with synaptogenesis during rat postnatal development.Neurochem. Res. 2008; 33: 1692-1700Crossref PubMed Scopus (21) Google Scholar; Lloyd-Burton and Roskams, 2012Lloyd-Burton S. Roskams A.J. SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS.J. Comp. Neurol. 2012; 520: 2575-2590Crossref PubMed Scopus (16) Google Scholar; Mendis et al., 1996Mendis D.B. Shahin S. Gurd J.W. Brown I.R. SC1, a SPARC-related glycoprotein, exhibits features of an ECM component in the developing and adult brain.Brain Res. 1996; 713: 53-63Crossref PubMed Scopus (37) Google Scholar; Risher et al., 2014Risher W.C. Patel S. Kim I.H. Uezu A. Bhagat S. Wilton D.K. Pilaz L.-J. Singh Alvarado J. Calhan O.Y. Silver D.L. et al.Astrocytes refine cortical connectivity at dendritic spines.Elife. 2014; 3: e04047Crossref Scopus (87) Google Scholar). However, hevin is also strongly expressed in many regions in adult brain in most astrocytes, and also in select populations of inhibitory and excitatory neurons (Hashimoto et al., 2016Hashimoto N. Sato T. Yajima T. Fujita M. Sato A. Shimizu Y. Shimada Y. Shoji N. Sasano T. Ichikawa H. SPARCL1-containing neurons in the human brainstem and sensory ganglion.Somatosens. Mot. Res. 2016; 33: 112-117Crossref PubMed Scopus (8) Google Scholar; Lively et al., 2007Lively S. Ringuette M.J. Brown I.R. Localization of the extracellular matrix protein SC1 to synapses in the adult rat brain.Neurochem. Res. 2007; 32: 65-71Crossref PubMed Scopus (31) Google Scholar; Lloyd-Burton and Roskams, 2012Lloyd-Burton S. Roskams A.J. SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS.J. Comp. Neurol. 2012; 520: 2575-2590Crossref PubMed Scopus (16) Google Scholar; Mendis et al., 1996Mendis D.B. Shahin S. Gurd J.W. Brown I.R. SC1, a SPARC-related glycoprotein, exhibits features of an ECM component in the developing and adult brain.Brain Res. 1996; 713: 53-63Crossref PubMed Scopus (37) Google Scholar; Mongrédien et al., 2019Mongrédien R. Erdozain A.M. Dumas S. Cutando L. Del Moral A.N. Puighermanal E. Rezai Amin S. Giros B. Valjent E. Meana J.J. et al.Cartography of hevin-expressing cells in the adult brain reveals prominent expression in astrocytes and parvalbumin neurons.Brain Struct. Funct. 2019; 224: 1219-1244Crossref PubMed Scopus (6) Google Scholar; Risher et al., 2014Risher W.C. Patel S. Kim I.H. Uezu A. Bhagat S. Wilton D.K. Pilaz L.-J. Singh Alvarado J. Calhan O.Y. Silver D.L. et al.Astrocytes refine cortical connectivity at dendritic spines.Elife. 2014; 3: e04047Crossref Scopus (87) Google Scholar). Like hevin, SPARC is broadly expressed during development in glial cells and radial glia (progenitor cells that additionally function as guide cells along which neurons migrate) (Vincent et al., 2008Vincent A.J. Lau P.W. Roskams A.J. SPARC is expressed by macroglia and microglia in the developing and mature nervous system.Dev. Dyn. 2008; 237: 1449-1462Crossref PubMed Scopus (53) Google Scholar), but in adult CNS, SPARC is expressed only in very limited regions by astrocytes and microglia but not neurons (Lloyd-Burton and Roskams, 2012Lloyd-Burton S. Roskams A.J. SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS.J. Comp. Neurol. 2012; 520: 2575-2590Crossref PubMed Scopus (16) Google Scholar; Mendis et al., 1995Mendis D.B. Malaval L. Brown I.R. SPARC, an extracellular matrix glycoprotein containing the follistatin module, is expressed by astrocytes in synaptic enriched regions of the adult brain.Brain Res. 1995; 676: 69-79Crossref PubMed Scopus (46) Google Scholar; Mongrédien et al., 2019Mongrédien R. Erdozain A.M. Dumas S. Cutando L. Del Moral A.N. Puighermanal E. Rezai Amin S. Giros B. Valjent E. Meana J.J. et al.Cartography of hevin-expressing cells in the adult brain reveals prominent expression in astrocytes and parvalbumin neurons.Brain Struct. Funct. 2019; 224: 1219-1244Crossref PubMed Scopus (6) Google Scholar; Vincent et al., 2008Vincent A.J. Lau P.W. Roskams A.J. SPARC is expressed by macroglia and microglia in the developing and mature nervous system.Dev. Dyn. 2008; 237: 1449-1462Crossref PubMed Scopus (53) Google Scholar). Given the high sequence identity, the striking biological differences between hevin and SPARC remain puzzling, especially as they also seem to share certain functions, as described above, and this dichotomy is important given the characteristic temporal and spatial expression patterns of hevin and SPARC. The molecular mechanisms of hevin and SPARC actions in the brain are unclear. In vivo, hevin appears to promote the interaction between two families of synaptic organizers, the post-synaptic neuroligins and the pre-synaptic neurexins, and it is proposed to join them together in transsynaptic bridges that span and stabilize synaptic junctions (Singh et al., 2016Singh S.K. Stogsdill J.A. Pulimood N.S. Dingsdale H. Kim Y.H. Pilaz L.-J. Kim I.H. Manhaes A.C. Rodrigues W.S. Pamukcu A. et al.Astrocytes assemble thalamocortical synapses by bridging NRX1α and NL1 via hevin.Cell. 2016; 164: 183-196Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Neurexin-neuroligin complexes are among the most extensively studied transsynaptic bridges and are particularly notable because of their diverse composition and their links to neuropsychiatric disorders (Rudenko, 2019Rudenko G. Neurexins—versatile molecular platforms in the synaptic cleft.Curr. Opin. Struct. Biol. 2019; 54: 112-121Crossref PubMed Scopus (15) Google Scholar; Südhof, 2017Südhof T.C. Synaptic neurexin complexes: a molecular code for the logic of neural circuits.Cell. 2017; 171: 745-769Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). In humans, five neuroligin (NLGN) genes encode proteins containing a membrane-tethered, dimeric ectodomain with a cholinesterase-like fold, which is variably diversified through alternative splicing at two splice insert sites, site A (SSA) and site B (SSB) (Bemben et al., 2015Bemben M.A. Shipman S.L. Nicoll R.A. Roche K.W. The cellular and molecular landscape of neuroligins.Trends Neurosci. 2015; 38: 496-505Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar; Südhof, 2017Südhof T.C. Synaptic neurexin complexes: a molecular code for the logic of neural circuits.Cell. 2017; 171: 745-769Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Similarly, three neurexin (NRXN) genes generate membrane-tethered proteins with highly variable extracellular regions. α-Neurexins contain 6 LNS (laminin G, neurexin, sex-hormone binding globule) domains and 3 EGF (epidermal growth factor)-like repeats, while β-neurexins are composed of only a single LNS domain. Neurexins are also extensively diversified at alternative splice sites (SS1–6) (Südhof, 2017Südhof T.C. Synaptic neurexin complexes: a molecular code for the logic of neural circuits.Cell. 2017; 171: 745-769Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Contrasting hevin as a promoter of neurexin-neuroligin transsynaptic bridges, the MDGA (MAM domain-containing glycosylphosphatidylinositol anchor protein) family of synaptic organizers (composed of six immunoglobulin [Ig] domains, one fibronectin III, and one MAM [meprin, A5 protein, PTPμ] domain tethered to the post-synaptic membrane) destabilizes neurexin-neuroligin transsynaptic bridges. They do so by binding to neuroligin and blocking the recruitment of neurexin (Elegheert et al., 2017Elegheert J. Cvetkovska V. Clayton A.J. Heroven C. Vennekens K.M. Smukowski S.N. Regan M.C. Jia W. Smith A.C. Furukawa H. et al.Structural mechanism for modulation of synaptic neuroligin-neurexin signaling by MDGA proteins.Neuron. 2017; 95: 896-913.e10Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar; Gangwar et al., 2017Gangwar S.P. Zhong X. Seshadrinathan S. Chen H. Machius M. Rudenko G. Molecular mechanism of MDGA1: regulation of neuroligin 2:neurexin trans-synaptic bridges.Neuron. 2017; 94: 1132-1141.e4Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar; Kim et al., 2017Kim J.A. Kim D. Won S.Y. Han K.A. Park D. Cho E. Yun N. An H.J. Um J.W. Kim E. et al.Structural insights into modulation of neurexin-neuroligin trans-synaptic adhesion by MDGA1/neuroligin-2 complex.Neuron. 2017; 94: 1121-1131.e6Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). But it is not known if, and how, MDGAs, hevin, and SPARC interplay. Furthermore, recent studies have drawn into question whether hevin interacts with the neurexin-neuroligin transsynaptic bridge directly, or even at all, and whether hevin's synaptogenic effect involves these transsynaptic bridges (Elegheert et al., 2017Elegheert J. Cvetkovska V. Clayton A.J. Heroven C. Vennekens K.M. Smukowski S.N. Regan M.C. Jia W. Smith A.C. Furukawa H. et al.Structural mechanism for modulation of synaptic neuroligin-neurexin signaling by MDGA proteins.Neuron. 2017; 95: 896-913.e10Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar; Gan and Südhof, 2020Gan K.J. Südhof T.C. SPARCL1 promotes excitatory but not inhibitory synapse formation and function independent of neurexins and neuroligins.J. Neurosci. 2020; 40: 8088-8102Crossref PubMed Scopus (16) Google Scholar). For SPARC, it is also not known if it binds neurexin and/or neuroligin. So, while hevin and SPARC are strategically positioned to impact neural circuitries in developing and mature brain, whether they carry out synergistic, opposing, or indeed independent functions, is not clear, in part due to the lack of structure-function relationships. Here, we establish interplay between the trio of proteins, hevin, SPARC, and MDGA, and elucidate the molecular mechanism. First, we show that the hevin FS-EC tandem has striking structural differences compared with SPARC, predominantly in the EC domain. We show that hevin and SPARC are both able to bind to neuroligins and neurexins, suggesting that they can act in concert to regulate neurexin-neuroligin transsynaptic bridges. We further show that the hevin FS domain, which is structurally conserved in SPARC, is sufficient to bind both neuroligin and neurexin with nanomolar affinity. Importantly, we find that hevin and MDGAs occupy overlapping binding sites on neuroligin, explaining how they compete with each other for neuroligin binding. Finally, we show that hevin, like SPARC, binds to collagen in solution, but we do not observe the same collagen-binding site as in the SPARC structure. Collectively, our results establish that hevin and SPARC can take part in a complex symphony of competing protein interactions, vying for synaptic organizers as well as interacting with extracellular matrix components. Key structural features provide rationales for both the shared and fundamentally different biological roles of hevin and SPARC in the brain. Hevin FS-EC and fragments were produced using baculovirus-mediated overexpression (Figures 1A and S1A). Because attempts to solve the hevin FS-EC X-ray crystal structure by molecular replacement using SPARC as a search model (PDB: 1BMO and 2V53) were unsuccessful, the Ca2+ ions in the EF-hands were substituted with holmium and the hevin crystal structure determined through single isomorphous replacement with anomalous scattering phasing (Table 1). The final model of hevin FS-EC was refined using data to 2.27 Å resolution (Rwork = 18.9%; Rfree = 24.1%) (Table 1). The hevin FS domain (V430-C509) is composed of an EGF-like repeat (V430-Q457) containing two disulfide bonds as well as a Kazal domain (D458-K509) containing a mixed α/β fold stabilized by three disulfide bonds (Figure 1B). The EC domain (C515-F664) houses two canonical helix-loop-helix EF-hands (EF1 and EF2) that each contain a Ca2+-binding site with seven-oxygen coordination; the EF-hands are connected by a short helical turn (D517FEV520) that packs adjacent to an elongated bundle of helices (αA, αBC, αC) (Figures 1B and 1C). A disulfide bond between C634 and C650 stabilizes EF2, with a second disulfide bond between C515 and C626 connecting EF1 to the linker (K510-T514) between the FS and EC domains. A large cleft separates the FS and EC domains. The interface between the two domains is small (∼200 Å2) and contains only a few residues from strand β5 of the FS domain and from helix αF and the loop connecting the two EF-hands in the EC domain (Figure 1B). Two hevin molecules (mol A and mol B) embrace each other tightly in the asymmetric unit of the crystal structure so that helices αA and αC from the EC domain of mol B interact with the EC domain and Kazal subdomain of the neighboring mol A, with Y539 from helix αA fitting like a key in the cleft; likewise, helices αA, and αBC from the EC domain of mol A insert into mol B in a similar manner (Figures 1D and S2A). Because mol A and mol B are stabilized by an additional Ca2+ ion binding between them, and the monomers adopt similarly “open” configurations between the FS and EC domains (Figures S2A and S2B), we tested whether the hevin FS-EC tandem behaves as a dimer in solution. However, in analytical ultracentrifugation, size-exclusion chromatography (SEC), and cross-linking studies, hevin FS-EC (hevin_C2) is monomeric in solution, even in an acidic buffer similar to that used for crystallization and/or in presence of increasing amounts of Ca2+ (Figures S2C–S2E). To gauge whether the FS and EC domains, which are kept apart in the crystal structure, are able to associate in solution, we used SEC and demonstrated that the isolated FS (hevin_C5) and EC (hevin_C6) domains do not associate in solution, at least not without the linker between the two domains (Figure 1E). Taken together, the hevin FS-EC tandem forms an elongated structure that exists as a monomer in solution, and the two domains appear independent of each other, suggesting that they can function autonomously.Table 1Data collection and refinement statisticsCrystalNative 1Native 2Holmium derivativeData collectionWavelength (Å)1.000011.000011.53532Space groupI212121I212121I212121Cell dimensions a, b, c (Å)57.66, 132.29, 149.2357.63, 131.78, 148.6257.75, 130.97, 148.00 α, β, γ (º)90, 90, 9090, 90, 9090, 90, 90Resolution (Å)33.00–2.27 (2.31–2.27)30.39–2.34 (2.38–2.34)43.00–3.41 (3.47–3.41)Mean I/σ(I)36.7 (1.9)40.0 (1.9)20.9 (1.0)Rmerge0.063 (1.266)0.062 (1.339)0.070 (0.889)Rpim0.019 (0.449)0.018 (0.490)0.029 (0.491)CC½0.998 (0.696)0.979 (0.672)0.972 (0.637)Unique reflections26,686 (1,309)23,942 (1,191)7,865 (322)Completeness (%)99.8 (99.1)99.9 (100.0)98.5 (85.2)Multiplicity12.7 (7.9)12.1 (8.3)6.7 (3.3)PhasingResolution (Å)–43.00–3.41No. of sites–5Bayes CC–49.7 ± 18.3Figure of merit–0.300RefinementResolution (Å)33.00–2.27 (2.36–2.27)Rwork/Rfree18.85/24.07 (22.75/31.33)Reflections used Rwork/Rfree25,958/1,298 (2,098/110)Non-hydrogen atoms3,636Protein3,403Water155Other78B factors (Å2), overall42.2 Protein42.3 Water37.9 Other50.4RMSD Bond lengths (Å)0.009 Bond angles (º)1.09Ramachandran plot (%) Favored97.3 Allowed2.7 Disallowed0.0Rotamer outliers, n (%)1 (0.27)CC, correlation coefficient; RMSD, root-mean-square deviation. Open table in a new tab CC, correlation coefficient; RMSD, root-mean-square deviation. The hevin FS-EC and SPARC FS-EC (Hohenester et al., 1997Hohenester E. Maurer P. Timpl R. Crystal structure of a pair of follistatin-like and EF-hand calcium-binding domains in BM-40.EMBO J. 1997; 16: 3778-3786Crossref PubMed Scopus (136) Google Scholar) structures feature striking differences despite their high level of sequence identity (56% for the FS domain and 63.3% for the EC domain, respectively) (Figures 2A and 2B ). Although the hevin and SPARC FS domain have similar folds (root-mean-square deviation [RMSD] = 1.2 Å for 79 Cα superimposable residues) (Figure 2C, left), the hevin FS domain is rotated away from the EC domain by ∼40º into an “open” form compared with SPARC (PDB: 1BMO) (Figure 2C, right). Consequently, the hevin FS-EC interface is smaller (∼200 Å2) compared with the SPARC FS-EC interface (∼370 Å2) even though most residues at the interface are conserved (Figure 2A). Unexpectedly, the hevin and SPARC EC domains show dramatic structural differences; only 94 Cα residues out of 128 (hevin) and 150 (SPARC) residues observed in the respective crystal structures superimpose with an RMSD of 1.5 Å (Figure 2C, right). The two EF-hands form a structurally conserved core, differing only in their connecting segment (Figure 2D). However, the region between the FS domain and EF-hand tandem, I512-N584 (I135-N223 in SPARC) is completely reorganized so that helices αA′, αA, αBC, and αC (here referred to as “variable helices”) now point away from the EF-hands (Figure 2D). Despite the high sequence identity (45 residues out of the 73-residue stretch I512-N584), the region containing these variable helices has completely rearranged: the single, contiguous helix αA in SPARC is broken up in hevin into two kinked helices (αA′-αA); helix αB in hevin is disordered; and helices αBC and αC adopt a different fold (Figure 2D). Interestingly, in SPARC, helices αA, αB, and αC play an important role in maintaining high-affinity Ca2+ binding of the EF-hand pair, in particular helix αA, a long amphiphilic helix that inserts into the hydrophobic core of the EF-hand pair (Hohenester et al., 1996Hohenester E. Maurer P. Hohenadl C. Timpl R. Jansonius J.N. Engel J. Structure of a novel extracellular Ca(2+)-binding module in BM-40.Nat. Struct. Biol. 1996; 3: 67-73Crossref PubMed Scopus (132) Google Scholar). Thus, despite significant sequence conservation, the hevin EC domain