N-terminal Module Research Articles

The structure of the N-terminal module of the cell wall hydrolase RipA and its role in regulating catalytic activity.

RipA plays a vital role during cell division of Mycobacterium tuberculosis by degrading the cell wall peptidoglycan at the septum, allowing daughter cell separation. The peptidoglycan degrading activity relies on the NlpC/P60 domain, and as it is potentially harmful when deregulated, spatial and temporal control is necessary in this process. The N-terminal domain of RipA has been proposed to play an inhibitory role blocking the C-terminal NlpC/P60 domain. Accessibility of the active site cysteine residue is however not limited by the presence of the N-terminal domain, but by the lid-module of the inter-domain linker, which is situated in the peptide binding groove of the crystal structures of the catalytic domain. The 2.2 Å resolution structure of the N-terminal domain, determined by Se-SAD phasing, reveals an all-α-fold with 2 long α-helices, and shows similarity to bacterial periplasmic protein domains with scaffold-building role. Size exclusion chromatography and SAXS experiments are consistent with dimer formation of this domain in solution. The SAXS data from the periplasmic two-domain RipA construct suggest a rigid baton-like structure of the N-terminal module, with the catalytic domain connected by a 24 residue long flexible linker. This flexible linker allows for a catalytic zone, which is part of the spatiotemporal control of peptidoglycan degradation.

The unique evolution of the carbohydrate-binding module CBM20 in laforin.

Laforin catalyses glycogen dephosphorylation. Mutations in its gene result in Lafora disease, a fatal progressive myoclonus epilepsy, the hallmark being water-insoluble, hyperphosphorylated carbohydrate inclusions called Lafora bodies. Human laforin consists of an N-terminal carbohydrate-binding module (CBM) from family CBM20 and a C-terminal dual-specificity phosphatase domain. Laforin is conserved in all vertebrates, some basal metazoans and a small group of protozoans. The present in silico study defines the evolutionary relationships among the CBM20s of laforin with an emphasis on newly identified laforin orthologues. The study reveals putative laforin orthologues in Trichinella, a parasitic nematode, and identifies two sequence inserts in the CBM20 of laforin from parasitic coccidia. Finally, we identify that the putative laforin orthologues from some protozoa and algae possess more than one CBM20.

Mechanism of early light signaling by the carboxy-terminal output module of Arabidopsis phytochrome B

Plant phytochromes are thought to transduce light signals by mediating the degradation of phytochrome-interacting transcription factors (PIFs) through the N-terminal photosensory module, while the C-terminal module, including a histidine kinase-related domain (HKRD), does not participate in signaling. Here we show that the C-terminal module of Arabidopsis phytochrome B (PHYB) is sufficient to mediate the degradation of PIF3 specifically and to activate photosynthetic genes in the dark. The HKRD is a dimerization domain for PHYB homo and heterodimerization. A D1040V mutation, which disrupts the dimerization of HKRD and the interaction between C-terminal module and PIF3, abrogates PHYB nuclear accumulation, photobody biogenesis, and PIF3 degradation. By contrast, disrupting the interaction between PIF3 and PHYB’s N-terminal module has little effect on PIF3 degradation. Together, this study demonstrates that the dimeric form of the C-terminal module plays important signaling roles by targeting PHYB to subnuclear photobodies and interacting with PIF3 to trigger its degradation.

Structure of mycobacterial 3'-to-5' RNA:DNA helicase Lhr bound to a ssDNA tracking strand highlights distinctive features of a novel family of bacterial helicases.

Mycobacterial Lhr is a DNA damage-inducible superfamily 2 helicase that uses adenosine triphosphate (ATP) hydrolysis to drive unidirectional 3′-to-5′ translocation along single-stranded DNA (ssDNA) and to unwind RNA:DNA duplexes en route. ATPase, translocase and helicase activities are encompassed within the N-terminal 856-amino acid segment. The crystal structure of Lhr-(1–856) in complex with AMPPNP•Mg2+ and ssDNA defines a new helicase family. The enzyme comprises two N-terminal RecA-like modules, a winged helix (WH) domain and a unique C-terminal domain. The 3′ ssDNA end binds in a crescent-shaped groove at the interface between the first RecA domain and the WH domain and tracks 5′ into a groove between the second RecA and C domains. A kissing interaction between the second RecA and C domains forms an aperture that demarcates a putative junction between the loading strand tail and the duplex, with the first duplex nucleoside bookended by stacking on Trp597. Intercalation of Ile528 between nucleosides of the loading strand creates another bookend. Coupling of ATP hydrolysis to RNA:DNA unwinding is dependent on Trp597 and Ile528, and on Thr145 and Arg279 that contact phosphates of the loading strand. The structural and functional data suggest a ratchet mechanism of translocation and unwinding coupled to ATP-driven domain movements.

CAMTA-Mediated Regulation of Salicylic Acid Immunity Pathway Genes in Arabidopsis Exposed to Low Temperature and Pathogen Infection.

Arabidopsis thaliana calmodulin binding transcription activator (CAMTA) factors repress the expression of genes involved in salicylic acid (SA) biosynthesis and SA-mediated immunity in healthy plants grown at warm temperature (22°C). This repression is overcome in plants exposed to low temperature (4°C) for more than a week and in plants infected by biotrophic and hemibiotrophic pathogens. Here, we present evidence that CAMTA3-mediated repression of SA pathway genes in nonstressed plants involves the action of an N-terminal repression module (NRM) that acts independently of calmodulin (CaM) binding to the IQ and CaM binding (CaMB) domains, a finding that is contrary to current thinking that CAMTA3 repression activity requires binding of CaM to the CaMB domain. Induction of SA pathway genes in response to low temperature did not occur in plants expressing only the CAMTA3-NRM region of the protein. Mutational analysis provided evidence that the repression activity of the NRM was suppressed by action of the IQ and CaMB domains responding to signals generated in response to low temperature. Plants expressing the CAMTA3-NRM region were also impaired in defense against the bacterial hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000. Our results indicate that the regulation of CAMTA3 repression activity by low temperature and pathogen infection involves related mechanisms, but with distinct differences.

HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33

SummaryInfection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine’s activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy.

Streptococcuspneumoniae surface protein PfbA is a versatile multidomain and multiligand-binding adhesin employing different binding mechanisms.

Streptococcuspneumoniae, one of the major human respiratory pathogens, uses its repertoire of surface proteins to adhere to the epithelium of the nasopharynx and lungs leading to colonization. PfbA is a conserved surface protein of S.pneumoniae and helps the bacterium to colonize the host by recognizing the extracellular matrix (ECM) molecule fibronectin, as well as blood proteins like plasminogen and human serum albumin. The crystal structure of rPfbA150-607 revealed it to possess a beta-helical region similar to those of carbohydrate-active enzymes as well as a C-terminal segment that resembles the fibronectin-binding regions of fibronectin-binding proteins. To get more insight into the putative carbohydrate-binding property of PfbA and its binding to various host molecules, we generated three different constructs of PfbA and characterized them by ELISA, isothermal titration calorimetry and bio-layer interferometry experiments. Importantly, the isothermal titration calorimetry experiments revealed that PfbA binds to different saccharides. Further, ELISA and bio-layer interferometry experiments identified that (a) apart from fibronectin and plasminogen, the beta helix of PfbA also binds to other ECM molecules (b) lysines are not responsible for PfbA's binding to plasminogen, (c) in comparison with native fibrinogen, deglycosylated-fibrinogen exhibits reduced binding affinity towards PfbA implying the importance of sugar molecule-PfbA interaction and (d) the C-terminal region of PfbA binds exclusively to the N-terminal F1 modules of fibronectin. Thus, the results of this study show PfbA to be a versatile multidomain and multiligand-binding protein employing different binding mechanisms. These results could be useful for structure-based designing of inhibitors against PfbA.

Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes

Enzymes known as bifunctional and bimodular prokaryotic type-I FAD synthetase (FADS) exhibit ATP:riboflavin kinase (RFK) and FMN:ATP adenylyltransferase (FMNAT) activities in their C-terminal and N-terminal modules, respectively, and produce flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These act as cofactors of a plethora of flavoproteins in all organisms. Therefore, regulation of their production maintains the cellular flavoproteome homeostasis. Here, we focus on regulation of the FMN synthesis in Corynebacterium ammoniagenes (Ca) by the inhibition of its RFK activity by substrates and products of the reaction. We use a truncated CaFADS variant consisting in the isolated C-terminal RFK module, whose RFK activity is similar to that of the full-length enzyme. Inhibition of the RFK activity by the RF substrate is independent of the FMNAT module, and FMN production, in addition to being inhibited by an excess of RF, is also inhibited by both of the reaction products. Pre-steady-state kinetic and thermodynamic studies reveal key aspects to the substrates induced fit to produce the catalytically competent complex. Among them, the role of Mg2+ in the concerted allocation of substrates for catalysis and the ensemble of non-competent complexes that contribute to the regulated inhibition of the RFK activity are particularly relevant.

The X-linked deubiquitinase USP9X is an integral component of centrosome

The X-linked deubiquitinase USP9X has been implicated in multiple pathological disorders including malignancies and X-linked intellectual disability. However, its biological function and substrate repertoire remain to be investigated. In this study, we utilized the tandem mass tag labeling assay to identify USP9X-regulated proteins and revealed that the expression of multiple genes is altered in USP9X-deficient cells. Interestingly, we showed that USP9X promotes stabilization of centrosome proteins PCM1 and CEP55 through its catalytic activity. Remarkably, we demonstrated that USP9X is physically associated and spatially co-localized with PCM1 and CEP55 in the centrosome, and we revealed that either PCM1 or CEP55 loss resulted in impairment of USP9X centrosome localization. Moreover, we showed that USP9X is required for centrosome duplication, and this effect is dependent on its catalytic activity and its N-terminal module, which is responsible for physical association of USP9X with PCM1 and CEP55. Collectively, our experiments identified USP9X as an integral component of the centrosome where it functions to stabilize PCM1 and CEP55 and promote centrosome biogenesis.

Tobacco etch virus protease mediating cleavage of the cellulose-binding module tagged colored proteins immobilized on the regenerated amorphous cellulose.

In this study, four fusion proteins were designed, in which the N-terminal cellulose-binding module as the affinity tag was immobilized on the regenerated amorphous cellulose (RAC), and the release of the C-terminal colored proteins was detected easily and rapidly after on-resin cleavage using the free tobacco etch virus protease (TEVp) variant, or the immobilized cognate protease with a binding capacity of up to 220mg protein per gram of RAC. The enhanced stability and repetitive use of the immobilized TEVp compensated slight loss of the catalytic efficiency toward the soluble protein substrate. On-resin cleavage and purity of the released target proteins are related to the context of the fusion tag, the incorporated linker composition, and the colored protein. Owing to low cost and high binding capacity of the RAC, the TEVp immobilized on the resin is an ideal alternative for removing fusion tag. The colored proteins are easily monitored in the on-resin process of fusion proteins, and rapid separation from RAC.

Intersubunit distances in full-length, dimeric, bacterial phytochrome Agp1, as measured by pulsed electron-electron double resonance (PELDOR) between different spin label positions, remain unchanged upon photoconversion

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.

Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2',3'-Phosphoesterase HD Domain and a C-Terminal 5'-OH Polynucleotide Kinase Domain.

5'- and 3'-end-healing reactions are key steps in nucleic acid break repair in which 5'-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3'-PO4 or 2',3'-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate the 5'-PO4 and 3'-OH termini required for sealing by classic polynucleotide ligases. End-healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2',3'-phosphoesterase HD domain and a C-terminal 5'-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5'-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2',3'-cyclic phosphate, RNA 3'-phosphate, RNA 2'-phosphate, and DNA 3'-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCE The present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5'- and 3'-end-healing enzymes that phosphorylate 5'-OH termini and dephosphorylate 2',3'-cyclic-PO4, 3'-PO4, and 2'-PO4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla.

БАКТЕРІАЛЬНА ЕКСПРЕСІЯ РЕКОМБІНАНТНОГО ФУНКЦІОНАЛЬНО АКТИВНОГО N-КІНЦЕВОГО МОДУЛЯ ТИРОЗИЛ-ТРНК СИНТЕТАЗИ B. TAURUS

The nucleotide sequence coding N-terminal module of Bos taurus tyrosyl-tRNA synthetase (mini TyrRS) was cloned into the bacterial expression vector pET23d(+). Bacterial expression of the recombinant protein mini TyrRS was performed in E. coli BL21 (DE3)pLysE cells with the use of the constructed vector pET-23d(+)39YRS for subsequent physical and chemical protein studies. The catalytic activity of the recombinant mini TyrRS has been studied in the aminoacylation reaction of homologous tRNATyr.

Белок бактериального нуклеоида Dps связывается со структурированными РНК

Architectural protein Dps of the bacterial nucleoid employs side groups of lysines at its N-terminal modules for interacting with the sugar-phosphate backbone of the DNA. Electrostatic nature of interaction assumes the potential ability of Dps to bind with any nucleotide sequence including RNA. The available data also indicate that Dps exhibits enhanced affinity to branched DNA structures. In RNA molecules such structures are formed more frequently than in DNA. Hence, the aim of this investigation was studying the ability of purified Dps immobilized on acrylate spheres to bind with short RNAs isolated from bacterial cells. It appeared that transport and small regulatory RNAs forming stable secondary structures are preferential targets for such interaction. Among RNAs identified in complexes with Dps 8 transcripts corresponded to intergenic spaces, which might indicate the presence of novel genes. Moreover, products 9-13 nucleotides long belonging to small untranslated RNAs SdsR and RyeA and transcribed from both strands of the same locus were registered. Since the number of longer transcripts from this region was at least five-fold lower, it can be presumed that two counter-synthesized products form a partly complementary duplex subjected to controlled processing. The selectivity of Dps to these molecules, as well as to other structured RNAs, indicates a possibility of its involvement not only in bacterial genome condensation, but also in maintaining the functional state of the transcriptome.

HLARP7 C-terminal domain contains an xRRM that binds the 3' hairpin of 7SK RNA.

The 7SK small nuclear ribonucleoprotein (snRNP) sequesters and inactivates the positive transcription elongation factor b (P-TEFb), an essential eukaryotic mRNA transcription factor. The human La-related protein group 7 (hLARP7) is a constitutive component of the 7SK snRNP and localizes to the 3′ terminus of the 7SK long noncoding RNA. hLARP7, and in particular its C-terminal domain (CTD), is essential for 7SK RNA stability and assembly with P-TEFb. The hLARP7 N-terminal La module binds and protects the 3′ end from degradation, but the structural and functional role of its CTD is unclear. We report the solution NMR structure of the hLARP7 CTD and show that this domain contains an xRRM, a class of atypical RRM first identified in the Tetrahymena thermophila telomerase LARP7 protein p65. The xRRM binds the 3′ end of 7SK RNA at the top of stem-loop 4 (SL4) and interacts with both unpaired and base-paired nucleotides. This study confirms that the xRRM is general to the LARP7 family of proteins and defines the binding site for hLARP7 on the 7SK RNA, providing insight into function.

The Crystal Structures of the N-terminal Photosensory Core Module of Agrobacterium Phytochrome Agp1 as Parallel and Anti-parallel Dimers

Agp1 is a canonical biliverdin-binding bacteriophytochrome from the soil bacterium Agrobacterium fabrum that acts as a light-regulated histidine kinase. Crystal structures of the photosensory core modules (PCMs) of homologous phytochromes have provided a consistent picture of the structural changes that these proteins undergo during photoconversion between the parent red light-absorbing state (Pr) and the far-red light-absorbing state (Pfr). These changes include secondary structure rearrangements in the so-called tongue of the phytochrome-specific (PHY) domain and structural rearrangements within the long α-helix that connects the cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) and the PHY domains. We present the crystal structures of the PCM of Agp1 at 2.70 Å resolution and of a surface-engineered mutant of this PCM at 1.85 Å resolution in the dark-adapted Pr states. Whereas in the mutant structure the dimer subunits are in anti-parallel orientation, the wild-type structure contains parallel subunits. The relative orientations between the PAS-GAF bidomain and the PHY domain are different in the two structures, due to movement involving two hinge regions in the GAF-PHY connecting α-helix and the tongue, indicating pronounced structural flexibility that may give rise to a dynamic Pr state. The resolution of the mutant structure enabled us to detect a sterically strained conformation of the chromophore at ring A that we attribute to the tight interaction with Pro-461 of the conserved PRXSF motif in the tongue. Based on this observation and on data from mutants where residues in the tongue region were replaced by alanine, we discuss the crucial roles of those residues in Pr-to-Pfr photoconversion.

Module recombination and functional integration of oligosaccharide-producing multifunctional amylase

The oligosaccharide-producing multifunctional amylase (OPMA-N) has both hydrolytic and transglycosyl activities. Our previous reports demonstrated that the function and catalytic versatility of OPMA-N is closely related to its oligomerization, and its oligomeric state is affected by several conserved residues, such as Trp358, and by the cooperation of its small, noncatalytic N-terminal module and the catalytic module. We have demonstrated that the residue Trp358 exposed on the surface of OPMA-N molecule and has an obvious impact on OPMA-N oligomerization mainly by the charge effects. In this study, we investigated the effects of module recombination on the functional integration of OPMA-N. A series of module recombinants of the N-terminal and catalytic modules revealed that the intramolecular, semi-intramolecular and semi-intermolecular interactions of the N-terminal module with two or more catalytic modules enhanced the substrate affinity of the catalytic modules and facilitated the transglycosyl activity and functional integration of the enzyme by mediating positive cooperativity between the catalytic modules. Free N-terminal module alone did not contribute to these effects. Based on the results of this study, we speculated that the substrate affinity, but not the maximal catalytic activity, was the primary driving force in the natural evolution of enzymes, and that molecules of natural or modular enzymes may have occupied some evolutionary spaces that could be expanded or exhumed through module recombination to increase their overall catalytic efficacies or eventual catalytic efficiencies. All of the results in this study could be applied to integration of enzyme function and for creation of novel enzymes. Additionally, our results may provide important insights into the evolution of enzymes or organisms.

Design, synthesis, and biological characterization of novel PEG-linked dimeric modulators for CXCR4

CXCR4 dimerization has been widely demonstrated both biologically and structurally. This paper mainly focused on the development of structure-based dimeric ligands that target CXCL12–CXCR4 interaction and signaling. This study presents the design and synthesis of a series of [PEG]n linked dimeric ligands of CXCR4 based on the knowledge of the homodimeric crystal structure of CXCR4 and our well established platform of chemistry and bioassays for CXCR4. These new ligands include [PEG]n linked homodimeric or heterodimeric peptides consisting of either two DV3-derived moieties (where DV3 is an all-d-amino acid analog of N-terminal modules of 1–10 (V3) residues of vMIP-II) or hybrids of DV3 moieties and CXCL121–8. Among a total of 24 peptide ligands, four antagonists and three agonists showed good CXCR4 binding affinity, with IC50 values of <50nM and <800nM, respectively. Chemotaxis and calcium mobilization assays with SUP-T1 cells further identified two promising lead modulators of CXCR4: ligand 4, a [PEG3]2 linked homodimeric DV3, was an effective CXCR4 antagonist (IC50=22nM); and ligand 21, a [PEG3]2 linked heterodimeric DV3–CXCL121–8, was an effective CXCR4 agonist (IC50=407nM). These dimeric CXCR4 modulators represent new molecular probes and therapeutics that effectively modulate CXCL12–CXCR4 interaction and function.

Allosteric communication between DNA-binding and light-responsive domains of diatom class I aureochromes.

The modular architecture of aureochrome blue light receptors, found in several algal groups including diatoms, is unique by having the LOV-type photoreceptor domain fused to the C-terminus of its putative effector, an N-terminal DNA-binding bZIP module. The structural and functional understanding of aureochromes’ light-dependent signaling mechanism is limited, despite their promise as an optogenetic tool. We show that class I aureochromes 1a and 1c from the diatom Phaeodactylum tricornutum are regulated in a light-independent circadian rhythm. These aureochromes are capable to form functional homo- and heterodimers, which recognize the ACGT core sequence within the canonical ‘aureo box’, TGACGT, in a light-independent manner. The bZIP domain holds a more folded and less flexible but extended conformation in the duplex DNA-bound state. FT-IR spectroscopy in the absence and the presence of DNA shows light-dependent helix unfolding in the LOV domain, which leads to conformational changes in the bZIP region. The solution structure of DNA bound to aureochrome points to a tilted orientation that was further validated by molecular dynamics simulations. We propose that aureochrome signaling relies on an allosteric pathway from LOV to bZIP that results in conformational changes near the bZIP-DNA interface without major effects on the binding affinity.

The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1.

H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation. We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.