Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

Efficient and cost effective production of active-form human PKB using silkworm larvae.

Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.

Structural insights into the recruitment of SMRT by the corepressor SHARP under phosphorylative regulation.

The transcriptional corepressors SMRT/NCoR, components of histone deacetylase complexes, interact with nuclear receptors and many other transcription factors. SMRT is a target for the ubiquitously expressed protein kinase CK2, which is known to phosphorylate a wide variety of substrates. Increasing evidence suggests that CK2 plays a regulatory role in many cellular events, particularly, in transcription. However, little is known about the precise mode of action involved. Here, we report the three-dimensional structure of a SMRT/HDAC1-associated repressor protein (SHARP) in complex with phosphorylated SMRT, as determined by solution NMR. Phosphorylation of the CK2 site on SMRT significantly increased affinity for SHARP. We also confirmed the significance of CK2 phosphorylation by reporter assay and propose a mechanism involving the process of phosphorylation acting as a molecular switch. Finally, we propose that the SPOC domain functions as a phosphorylation binding module.

Microtubule-binding sites of the CH domain of EB1 and its autoinhibition revealed by NMR.

End-binding protein 1 (EB1) is one of the best studied plus-end tracking proteins. It is known that EB1 specifically binds the plus ends of microtubules (MTs) and promotes MT growth. EB1 activity is thought to be autoinhibited by an intramolecular interaction. Recent cryo-EM analyses showed that the CH domain of Mal3p (Schizosaccharomyces pombe EB1 homolog) binds to GMPCPP-MT (Sandblad, L. Cell 127 (2006) 1415-24), and strongly binds GTPγS-MT which is proposed to mimic MT plus ends better than GMPCPP-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Here, we report on the MT binding sites of the CH domain of EB1 as revealed by NMR using the transferred cross-saturation method. In this study, we used GMPCPP-MT and found that the MT binding sites are very similar to the binding site for GTPγS-MT as suggested by cryo-EM (Maurer S.P. et al. Cell 149 (2012) 371-82). Notably, the N-terminal tip of helix α6 of the CH domain did not make contact with GMPCPP-MT, in contrast to the cryo-EM study which showed that it is closely located to a putative switch region of ß-tubulin in GTPγS-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Further, we found that the intramolecular interaction site of EB1 overlaps the MT binding sites, indicating that the MT binding sites are masked by interaction with the C-terminal domain. We propose a structural view of autoinhibition and its release mechanism through competition binding with binding partners such as adenomatous polyposis coli protein.

Structural basis for CD44 recognition by ERM proteins.

CD44 is an important adhesion molecule that functions as the major hyaluronan receptor which mediates cell adhesion and migration in a variety of physiological and pathological processes. Although full activity of CD44 requires binding to ERM (ezrin/radixin/moesin) proteins, the CD44 cytoplasmic region, consisting of 72 amino acid residues, lacks the Motif-1 consensus sequence for ERM binding found in intercellular adhesion molecule (ICAM)-2 and other adhesion molecules of the immunoglobulin superfamily. Ultracentrifugation sedimentation studies and circular dichroism measurements revealed an extended monomeric form of the cytoplasmic peptide in solution. The crystal structure of the radixin FERM domain complexed with a CD44 cytoplasmic peptide reveals that the KKKLVIN sequence of the peptide forms a beta strand followed by a short loop structure that binds subdomain C of the FERM domain. Like Motif-1 binding, the CD44 beta strand binds the shallow groove between strand beta5C and helix alpha1C and augments the beta sheet beta5C-beta7C from subdomain C. Two hydrophobic CD44 residues, Leu and Ile, are docked into a hydrophobic pocket with the formation of hydrogen bonds between Asn of the CD44 short loop and loop beta4C-beta5C from subdomain C. This binding mode resembles that of NEP (neutral endopeptidase 24.11) rather than ICAM-2. Our results reveal a characteristic versatility of peptide recognition by the FERM domains from ERM proteins, suggest a possible mechanism by which the CD44 tail is released from the cytoskeleton for nuclear translocation by regulated intramembrane proteolysis, and provide a structural basis for Smad1 interactions with activated CD44 bound to ERM protein.

Structural basis of the cytoplasmic tail of adhesion molecule CD43 and its binding to ERM proteins.

CD43/leukosialin/sialophorin is the major adhesion molecule in most hematopoietic cells and belongs to the sialomucin superfamily. In leukocyte emigration and activation, the exclusion of CD43 from the immunological synapse is an essential step. While the exclusion requires binding of the cytoplasmic region to ERM (ezrin/radixin/moesin) proteins, the detailed specific nature of the interaction between CD43 and ERM proteins is obscure. We have characterized the conformational properties of the CD43 cytoplasmic region, consisting of 124 amino acid residues, by hydrodynamic and spectroscopic measurements. Sedimentation equilibrium and velocity studies of ultracentrifugation revealed that the CD43 cytoplasmic peptide exists in a monomeric and extended form in solution. The crystal structure of the complex between the radixin FERM (4.1 and ERM) domain and the CD43 juxtamembrane region peptide reveals that the nonpolar region of the peptide binds subdomain C of the FERM domain. CD43 lacks the Motif-1 sequence for FERM binding found in the FERM-intercellular adhesion molecule-2 complex but possesses two conserved leucine residues that dock into the hydrophobic pocket of subdomain C without forming a 3(10)-helix. The FERM-binding site on CD43 is overlapped with the functional nuclear localization signal sequence. Our structure suggests that regulation of ERM binding may be coupled with regulated intramembrane proteolysis of CD43 followed by the nuclear transfer of the cytoplasmic peptide.

Structural basis of PSGL-1 binding to ERM proteins.

P-selectin glycoprotein ligand-1 (PSGL-1), an adhesion molecule with O-glycosylated extracellular sialomucins, is involved in leukocyte inflammatory responses. On activation, ezrin-radixin-moesin (ERM) proteins mediate the redistribution of PSGL-1 on polarized cell surfaces to facilitate binding to target molecules. ERM proteins recognize a short binding motif, Motif-1, conserved in cytoplasmic tails of adhesion molecules, whereas PSGL-1 lacks Motif-1 residues important for binding to ERM proteins. The crystal structure of the complex between the radixin FERM domain and a PSGL-1 juxtamembrane peptide reveals that the peptide binds the groove of FERM subdomain C by forming a beta-strand associated with strand beta5C, followed by a loop flipped out towards the solvent. The Motif-1 3(10) helix present in the FERM-ICAM-2 complex is absent in PSGL-1 given the absence of a critical Motif-1 alanine residue, and PSGL-1 reduces its contact area with subdomain C. Non-conserved positions are occupied by large residues Met9 and His8, which stabilize peptide conformation and enhance groove binding. Non-conserved residues play an important role in compensating for loss of binding energy resulting from the absence of conserved residues important for binding.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of adhesion molecule CD44.

CD44 is an important adhesion molecule that specifically binds hyaluronic acid and regulates cell-cell and cell-matrix interactions. Increasing evidence has indicated that CD44 is assembled in a regulated manner into the membrane-cytoskeletal junction, a process that is mediated by ERM (ezrin/radixin/moesin) proteins. Crystals of a complex between the radixin FERM domain and the C-terminal cytoplasmic region of CD44 have been obtained. The crystal of the radixin FERM domain bound to the CD44 cytoplasmic tail peptide belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.70, b = 66.18, c = 86.22 A, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.1 A.

Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition.

Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus end-tracking proteins that regulate microtubule dynamics, but it is obscure how CLIP-170 recognizes the microtubule plus end and contributes to polymerization rescue. Crystallographic, NMR, and mutation studies of two tandem cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170, CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a more basic groove and directly binds the EExEEY/F motif of the C-terminal acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic tails. These results provide a structural basis for the proposed CLIP-170 copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating that EB1 localized at the plus end contributes to CLIP-170 recruitment to the plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or nucleation by neutralizing the negative charges of tubulins with the highly positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds microtubule, the released zinc knuckle domain may serve to recruit dynein to the plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tails of adhesion molecules CD43 and PSGL-1.

Radixin is a member of the ERM proteins that cross-link plasma membranes and actin filaments. The FERM domains located in the N-terminal regions of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. Here, crystals of the radixin FERM domain bound to the cytoplasmic peptides of two adhesion molecules, CD43 and PSGL-1, have been obtained. Crystals of the radixin FERM domain bound to CD43 belong to space group P4(3)22, with unit-cell parameters a = b = 68.72, c = 201.39 A, and contain one complex in the crystallographic asymmetric unit. Crystals of the radixin FERM domain bound to PSGL-1 belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 80.74, b = 85.73, c = 117.75 A, and contain two complexes in the crystallographic asymmetric unit. Intensity data sets were collected to a resolution of 2.9 A for the FERM-CD43 complex and 2.8 A for the FERM-PSGL-1 complex.

Structural basis for NHERF recognition by ERM proteins.

The Na+/H+ exchanger regulatory factor (NHERF) is a key adaptor protein involved in the anchoring of ion channels and receptors to the actin cytoskeleton through binding to ERM (ezrin/radixin/moesin) proteins. NHERF binds the FERM domain of ERM proteins, although NHERF has no signature Motif-1 sequence for FERM binding found in adhesion molecules. The crystal structures of the radixin FERM domain complexed with the NHERF-1 and NHERF-2 C-terminal peptides revealed a peptide binding site of the FERM domain specific for the 13 residue motif MDWxxxxx(L/I)Fxx(L/F) (Motif-2), which is distinct from Motif-1. This Motif-2 forms an amphipathic alpha helix for hydrophobic docking to subdomain C of the FERM domain. This docking causes induced-fit conformational changes in subdomain C and affects binding to adhesion molecule peptides, while the two binding sites are not overlapped. Our studies provide structural paradigms for versatile ERM linkages between membrane proteins and the cytoskeleton.

Structural basis of the Rho GTPase signaling.

Small GTPases of the Rho family serve as conformational switches in a wide variety of signal transduction pathways that regulate diverse cellular functions. The GTP-bound forms of Rho GTPases are capable of interacting with downstream effectors that control cytoskeletal rearrangements. Regulators that stimulate nucleotide exchange, the hydrolytic cycle and distribution between the membrane and cytosol control the switch. Detailed pictures of Rho GTPase switching, effector recognition and regulation by regulators have emerged from recent structural investigations. These include the most extensively studied Rho GTPases, RhoA, Rac1, 2 and Cdc42, and their complexes with effectors and regulators. These studies have revealed the general diversity of effector and regulator structures, and in particular the structural features concerning the specific interactions involved in Rho effector recognition and regulator interactions with Rho GTPase. These findings provide a critical insight into the nature of Rho GTPase activity and consequently allow for a detailed manipulation of signaling pathways mediated by these proteins.

Parallel coiled-coil association of the RhoA-binding domain in Rho-kinase.

Rho-kinase is a serine/threonine protein kinase that regulates cytoskeletal events in cells. The enzyme activity of Rho-kinase is auto-inhibited in the free state but is activated through direct binding to the small GTPase Rho in the GTP-bound form. The crystal structure of the Rho-binding domain (RhoBD) of Rho-kinase has been determined at 1.8-A resolution by the multi-wavelength anomalous dispersion technique. The structure shows that RhoBD dimerizes to form a parallel coiled-coil with long consecutive alpha-helices extended to approximately 97 A and suggests that free Rho-kinase can also form a dimer through parallel self-association. At the middle region of the coiled-coil, the polypeptide chains are flexible and display loose "knobs-into-holes" packing of the side chains from both chains. RhoBD residues that have been shown to be critical for Rho-binding are spread in the positively charged C-terminal region. The parallel coiled-coil structure of our Rho-kinase RhoBD in the free form is different from the anti-parallel coiled-coil structure of RhoBD of protein kinase N when complexed with RhoA. Implications derived from these structural studies in relation to the mechanism of Rho-kinase activation will be addressed with previously reported experimental data.

Crystallographic characterization of the radixin FERM domain bound to the C-terminal region of the human Na+/H+-exchanger regulatory factor (NHERF).

Radixin is a member of the ERM proteins, which cross-link plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins interact with Na(+)/H(+)-exchanger regulatory factors (NHERFs), which are PDZ-containing adaptor proteins, to modulate the ion-channel activity. Here, crystals of complexes between the radixin FERM domain and the C-terminal regions of NHERF and NHERF2 have been obtained. The crystals of the FERM-NHERF complex were found to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 69.38 (2), b = 146.27 (4), c = 177.76 (7) A. The crystal contains four complexes in the asymmetric unit. An intensity data set was collected to a resolution of 2.50 A.