Mechanism of action of a Janus-faced single-domain protein inhibitor simultaneously targeting two peptidase classes

Protein inhibitors provide a physiological mechanism for the regulation of proteolytic enzymes. While most single-domain inhibitors have one reactive site with which they target peptidases of a specific catalytic class, selected specimens inhibit two peptidase molecules simultaneously, thus giving rise to ternary complexes. To study such inhibition, we analyzed the function of one of these proteins, sermetstatin, which strongly binds as a dimer to serine proteinases (SPs) and a metallopeptidase (MP). In addition, we determined the structures of the isolated inhibitor dimer and its heterotetrameric complexes with the SP subtilisin and the MP snapalysin, which reveal that inhibition occurs through two independent distal reactive sites. These structures and the derived model for the heterohexameric complex provide for the first time a detailed view of the molecular mechanism of simultaneous inhibition of proteinases belonging to two distinct mechanistic classes by a single-domain protein. © The Royal Society of Chemistry 2013.Peer Reviewe

Structure of the catalytic domain of the Tannerella forsythia matrix metallopeptidase karilysin in complex with a tetrapeptidic inhibitor

5 páginas, 1 figura, 1 tabla.-- et al.Karilysin is the only metallopeptidase identified as a virulence factor in the odontopathogen Tannerella forsythia owing to its deleterious effect on the host immune response during bacterial infection. The very close structural and sequence-based similarity of its catalytic domain (Kly18) to matrix metalloproteinases suggests that karilysin was acquired by horizontal gene transfer from an animal host. Previous studies by phage display identified peptides with the consensus sequence XWFPXXXGGG (single-letter amino-acid codes; X represents any residue) as karilysin inhibitors with low-micromolar binding affinities. Subsequent refinement revealed that inhibition comparable to that of longer peptides could be achieved using the tetrapeptide SWFP. To analyze its binding, the high-resolution crystal structure of the complex between Kly18 and SWFP was determined and it was found that the peptide binds to the primed side of the active-site cleft in a substrate-like manner. The catalytic zinc ion is clamped by the α-amino group and the carbonyl O atom of the serine, thus distantly mimicking the general manner of binding of hydroxamate inhibitors to metallopeptidases and contributing, together with three zinc-binding histidines from the protein scaffold, to an octahedral-minus-one metal-coordination sphere. The tryptophan side chain penetrates the deep partially water-filled specificity pocket of Kly18. Together with previous serendipitous product complexes of Kly18, the present results provide the structural determinants of inhibition of karilysin and open the field for the design of novel inhibitory strategies aimed at the treatment of human periodontal disease based on a peptidic hit molecule. © 2013.This study was supported in part by grants from European, American, Polish, Spanish, Danish and Catalan agencies (2012/04/A/NZ1/00051, 2011/03/N/NZ1/00586, 2137/7.PR-EU/2011/2, DE09761, FP7-HEALTH-F3-2009-223101 ‘AntiPathoGN’, FP7-HEALTH-2010-261460 ‘Gums&Joints’, FP7-PEOPLE-2011-ITN-290246 ‘RAPID’, BIO2009-10334, BFU2012-32862, CSD2006-00015, Lundbeck Foundation grant R54-A5291 and Fundació ‘La Marató de TV3’ grants 2009-100732 and 2009SGR1036). The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University in Kraków (Poland) is a beneficiary of structural funds from the European Union (grant No POIG.02.01.00-12-064/08 ‘Molecular Biotechnology for Health’).Peer Reviewe

Estudio estructural y funcional de un inhibidor proteínico monodominio de doble faz, sermetstatina, en complejo con dos peptidasas de diferente clase, subtilisina y esnapalisina

Los inhibidores de peptidasas representan un mecanismo fisiológico para la regulación de las enzimas proteolíticas. Mientras que mayoría de los inhibidores monodominio tienen un único sitio reactivo mediante el cual interaccionan con sus peptidasas dianas de un tipo catalítico específico, algunos de ellos inhiben dos moléculas de peptidasa simultáneamente, dando lugar a la formación de complejos ternarios. Para estudiar este tipo de inhibidores se analizó la función de uno de ellos, sermetstatina. Este inhibidor forma un dímero que une fuertemente serín peptidasas y metalopeptidasas. La estructura del dímero de inhibidor fue determinada revelando que sermetstatina presenta una conformación en α/β-sándwich alargado constituido por cinco hebras β antiparalelas conectadas entre sí (β3-β2-β1-β4-β5; conectividad -1, -1, +3, +1) que dan lugar a una hoja β girada ∼30o, en cuya cara cóncava se acomodan dos hélices α (α1 y α2) y una hélice 310. Además, las estructuras de los complejos heterotetraméricos de sermetstatina con la serín peptidasa subtilisina y la metalopeptidasa esnapalisina fueron también determinadas, mostrando que la inhibición ocurre a través de lazos reactivos distales independientes. La interacción entre subtilisina y sermetstatina se produce mediante el lazo reactivo 2, posicionado adecuadamente por su hélice de anclaje, y la hendidura del centro activo de la enzima. El lazo se inserta a modo de cuña mimetizando un substrato en conformación extendida y canónica en la hendidura del centro activo de la enzima, siguiendo el mecanismo estándar de inhibición. Por otro lado, la interacción entre esnapalisina y sermetstatina se produce mediante el extremo N-terminal, el lazo reactivo 1, la hélice α1 y la región Lβ4β5 del inhibidor; y la hendidura del centro activo de la enzima y exositios presentes en la superficie de la proteasa. Este modo de inhibición sigue un mecanismo novedoso en inhibidores de metalopeptidasas, siendo este una reminiscencia distante del modo inhibitorio de los TIMPs en su unión con MMPs así como del modo inhibitorio del inhibidor de serralisina sobre la metalopeptidasa serralisina. Estas estructuras y el modelo del complejo heterohexamérico proporcionan por primera vez una visión detallada del mecanismo molecular de la inhibición simultánea de peptidasas pertenecientes a dos clases mecanísticamente diferentes por un inhibidor monodominio. En resumen, en el presente trabajo se ha determinado que sermetstatina es un inhibidor de doble faz genuino monodominio que ha evolucionado a partir de inhibidores de serín peptidasas de la familia MEROPS I16 con un único sitio reactivo que siguen el mecanismo estándar de inhibición. Dicha evolución ha dado lugar a una proteína bifuncional capaz de inhibir simultáneamente diferentes serín peptidasas y una metalopeptidasa específica a través de dos sitios reactivos distales compatibles.Protein inhibitors provide a physiological mechanism for the regulation of proteolytic enzymes. While most single-domain inhibitors have one reactive site with which they target peptidases of a specific catalytic class, selected specimens inhibit two peptidase molecules simultaneously, thus giving rise to ternary complexes. To study such inhibition, the function of one of these proteins, sermetstatin, was analyzed. This inhibitor strongly binds as a dimer to serine peptidases and a metallopeptidase. The structure of the isolated inhibitor dimer was determined revealing that sermetstatin is an elongated α/β-sandwich. It consists of a five-stranded antiparallel β-sheet (β3-β2-β1-β4-β5; connectivity -1,-1,+3,+1) twisted by ∼30o, whose concave face accommodates two α-helices (α1 and α2) and a 310-helix. In addition, the structures of the heterotetrameric complexes with the serine peptidase subtilisin and the metallopeptidase snapalysin were equally determined, showing that inhibition occurs through two independent distal reactive sites. The subtilisin-sermetstatin interaction is made by reactive-site loop 2, adequately positioned by its scaffold helix, and the active-site cleft of the enzyme. The loop is inserted wedge-like mimicking a substrate in extended, “canonical” conformation in the active-site cleft of the enzyme following the “standard mechanism”. On the other hand, the snapalysin-sermetstatin interaction involves the N-terminal tail, reactive site loop 1, helix α1 and Lβ4β5 of the inhibitor; and the active-site cleft of the enzyme and some exosites on the protease surface. This inhibition modus follows a novel mechanism for metallopeptidase inhibitors only distantly reminiscent of the inhibitory mode of tissue inhibitors of metalloproteinases on their target matrix metalloproteinases and of serralysin inhibitors on their cognate serralysin MPs. These structures and the derived model for the heterohexameric complex provide for the first time a detailed view of the molecular mechanism of simultaneous inhibition of proteinases belonging to two distinct mechanistic classes by a single-domain protein. In summary, it was determined that sermetstatin is a genuine Janus-faced single-domain inhibitor which has evolved from single-site standard-mechanism serine peptidase inhibitors of family I16 to give a protein capable of simultaneous inhibition of serine peptidase in general and a specific metallopeptidase through distinct but compatible sites

Estudio estructural y funcional de un inhibidor proteínico monodominio de doble faz, sermetstatina, en complejo con dos peptidasas de diferente clase, subtilisina y esnapalisina

Peer Reviewe

Functional and structural insights into astacin metallopeptidases

The astacins are a family of multi-domain metallopeptidases with manifold functions in metabolism. They are either secreted or membrane-anchored and are regulated by being synthesized as inactive zymogens and also by colocalizing protein inhibitors. The distinct family members consist of N-terminal signal peptides and pro-segments, zincdependent catalytic domains, further downstream extracellular domains, transmembrane anchors, and cytosolic domains. The catalytic domains of four astacins and the zymogen of one of these have been structurally characterized and shown to comprise compact ~200-residue zinc-dependent moieties divided into an N-terminal and a C-terminal sub-domain by an active-site cleft. Astacins include an extended zinc-binding motif (HEXXHXXGXXH) which includes three metal ligands and groups them into the metzincin clan of metallopeptidases. In mature, unbound astacins, a conserved tyrosine acts as an additional zinc ligand, which is swung out upon substrate or inhibitor binding in a 'tyrosine switch' motion. Other characteristic structural elements of astacin catalytic domains are three large α-helices and a five-stranded β-sheet, as well as two or three disulfi de bonds. The N-terminal pro-segments are variable in length and rather unstructured. They inhibit the catalytic zinc following an 'aspartate-switch' mechanism mediated by an aspartate embedded in a conserved motif (FXGD). Removal of the pro-segment uncovers a deep and extended active-site cleft, which in general shows preference for aspartate residues in the specifi city pocket (S 1′). Furthermore, astacins undergo major rearrangement upon activation within an 'activation domain,' and show a slight hinge movement when binding substrates or inhibitors. In this review, we discuss the overall architecture of astacin catalytic domains and their involvement in function and zymogenic activation. Copyright © 2011-2012 by Walter de Gruyter.Financial support was provided by grants from European, Spanish, German, and Catalan agencies [FP7-HEALTH-F3-2009-223101 ‘ AntiPathoGN, ’FP7-HEALTH-2010-261460 ‘ Gums & Joints, ’ FP7-PEOPLE-2011-290246 ‘ RAPID, ’ BIO2009-10334, CSD2006-00015, 2009SGR1036, Fundació La Marató de TV3 100372, DFG Graduate School 1043 Immunotherapy; Natural Sciences and Medical Research Center (NMFZ) Mainz]. S.T.-M. is the recipient of an FPI fellowship from the former Spanish Ministry for Science and Technology.Peer Reviewe

Ultratight crystal packing of a 10 kDa protein

7 páginas, 2 figuras, 2 tablas.-- et al.While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported. © 2013 International Union of Crystallography Printed in Singapore - all rights reserved.This study was supported in part by grants from the American, European, Lithuanian, Spanish and Catalan agencies (FP7-HEALTHF3-2009-223101 ‘AntiPathoGN’, FP7-HEALTH-2010-261460 ‘Gums & Joints’, FP7-PEOPLE-2011-ITN-290246 ‘RAPID’, 31V-151 ‘COSMETIZYM’, BIO2009-10334, BFU2009-07134/BMC, BFU2012-32862, BFU2012-33516, CSD2006-00015, a JAE postdoctoral contract from CSIC, an FPU PhD fellowship from the Spanish Ministry for Science and Technology and Fundació ‘La Marató de TV3’ grants 2009-100732 and 2009SGR1036).Peer Reviewe

Estudio estructural y funcional de un inhibidor proteínico monodominio de doble faz, sermetstatina, en complejo con dos peptidasas de diferente clase, subtilisina y esnapalisina

A portada: Universitat Autònoma de Barcelona, Institut de Biotecnologia i Biomedicina: Consejo Superior de Investigaciones Científicas: Institut de Biologia Molecular de Barcelona, Departament de Biologia Estructural, Proteolysis LaboratoryLos Inhibidores de peptidasas representan un mecanismo fisiológico para la regulación de las enzimas proteolíticas. Mientras que mayoría de los inhibidores monodominio tienen un único sitio reactivo mediante el cual interaccionan con sus peptidasas dianas de un tipo catalítico específico, algunos de ellos inhiben dos moléculas de peptidasa simultáneamente, dando lugar a la formación de complejos ternarios. Para estudiar este tipo de inhibidores se analizó la función de uno de ellos, sermetstatina. Este inhibidor forma un dímero que une fuertemente serín peptidasas y metalopeptidasas. La estructura del dímero de inhibidor fue determinada revelando que sermetstatina presenta una conformación en α/β-sándwich alargado constituido por cinco hebras β antiparalelas conectadas entre sí (β3-β2-β1-β4-β5; conectividad -1, -1, +3, +1) que dan lugar a una hoja β girada ∼30o, en cuya cara cóncava se acomodan dos hélices α (α1 y α2) y una hélice 310. Además, las estructuras de los complejos heterotetraméricos de sermetstatina con la serín peptidasa subtilisina y la metalopeptidasa esnapalisina fueron también determinadas, mostrando que la inhibición ocurre a través de lazos reactivos distales independientes. La interacción entre subtilisina y sermetstatina se produce mediante el lazo reactivo 2, posicionado adecuadamente por su hélice de anclaje, y la hendidura del centro activo de la enzima. El lazo se inserta a modo de cuña mimetizando un substrato en conformación extendida y canónica en la hendidura del centro activo de la enzima, siguiendo el mecanismo estándar de inhibición. Por otro lado, la interacción entre esnapalisina y sermetstatina se produce mediante el extremo N-terminal, el lazo reactivo 1, la hélice α1 y la región Lβ4β5 del inhibidor; y la hendidura del centro activo de la enzima y exositios presentes en la superficie de la proteasa. Este modo de inhibición sigue un mecanismo novedoso en inhibidores de metalopeptidasas, siendo este una reminiscencia distante del modo inhibitorio de los TIMPs en su unión con 07Ps así como del modo inhibitorio del inhibidor de serralisina sobre la metalopeptidasa serralisina. Estas estructuras y el modelo del complejo heterohexamérico proporcionan por primera vez una visión detallada del mecanismo molecular de la inhibición simultánea de peptidasas pertenecientes a dos clases mecanísticamente diferentes por un inhibidor monodominio. En resumen, en el presente trabajo se ha determinado que sermetstatina es un inhibidor de doble faz genuino monodominio que ha evolucionado a partir de inhibidores de serín peptidasas de la familia MEROPS I16 con un único sitio reactivo que siguen el mecanismo estándar de inhibición. Dicha evolución ha dado lugar a una proteína bifuncional capaz de inhibir simultáneamente diferentes serín peptidasas y una metalopeptidasa específica a través de dos sitios reactivos distales compatibles.Protein inhibitors provide a physiological mechanism for the regulation of proteolytic enzymes. While most single-domain inhibitors have one reactive site with which they target peptidases of a specific catalytic class, selected specimens inhibit two peptidase molecules simultaneously, thus giving rise to ternary complexes. To study such inhibition, the function of one of these proteins, sermetstatin, was analyzed. This inhibitor strongly binds as a dimer to serine peptidases and a metallopeptidase. The structure of the isolated inhibitor dimer was determined revealing that sermetstatin is an elongated α/β-sandwich. It consists of a five-stranded antiparallel β-sheet (β3-β2-β1-β4-β5; connectivity -1,-1,+3,+1) twisted by ∼30o, whose concave face accommodates two α-helices (α1 and α2) and a 310-helix. In addition, the structures of the heterotetrameric complexes with the serine peptidase subtilisin and the metallopeptidase snapalysin were equally determined, showing that inhibition occurs through two independent distal reactive sites. The subtilisin-sermetstatin interaction is made by reactive-site loop 2, adequately positioned by its scaffold helix, and the active-site cleft of the enzyme. The loop is inserted wedge-like mimicking a substrate in extended, "canonical" conformation in the active-site cleft of the enzyme following the "standard mechanism". On the other hand, the snapalysin-sermetstatin interaction involves the N-terminal tail, reactive site loop 1, helix α1 and Lβ4β5 of the inhibitor; and the active-site cleft of the enzyme and some exosites on the protease surface. This inhibition modus follows a novel mechanism for metallopeptidase inhibitors only distantly reminiscent of the inhibitory mode of tissue inhibitors of metalloproteinases on their target matrix metalloproteinases and of serralysin inhibitors on their cognate serralysin MPs. These structures and the derived model for the heterohexameric complex provide for the first time a detailed view of the molecular mechanism of simultaneous inhibition of proteinases belonging to two distinct mechanistic classes by a single-domain protein. In summary, it was determined that sermetstatin is a genuine Janus-faced single-domain inhibitor which has evolved from single-site standard-mechanism serine peptidase inhibitors of family I16 to give a protein capable of simultaneous inhibition of serine peptidase in general and a specific metallopeptidase through distinct but compatible sites

The intestinal MUC2 mucin C-terminus is stabilized by an extra disulfide bond in comparison to von Willebrand factor and other gel-forming mucins

The MUC2 mucin is a large, highly glycosylated polymer that builds the intestinal mucus. Here, the authors generate a high-resolution structural model of the 800 amino acid C-terminal dimer including its glycans. Stabilization is achieved by interdimer disulfide-bonds in both ends, essential for a stable mucus barrier

Structural mechanism of signal transduction in a phytochrome histidine kinase

Publisher Copyright: © 2022, The Author(s).Phytochrome proteins detect red/far-red light to guide the growth, motion, development and reproduction in plants, fungi, and bacteria. Bacterial phytochromes commonly function as an entrance signal in two-component sensory systems. Despite the availability of three-dimensional structures of phytochromes and other two-component proteins, the conformational changes, which lead to activation of the protein, are not understood. We reveal cryo electron microscopy structures of the complete phytochrome from Deinoccocus radiodurans in its resting and photoactivated states at 3.6 Å and 3.5 Å resolution, respectively. Upon photoactivation, the photosensory core module hardly changes its tertiary domain arrangement, but the connector helices between the photosensory and the histidine kinase modules open up like a zipper, causing asymmetry and disorder in the effector domains. The structures provide a framework for atom-scale understanding of signaling in phytochromes, visualize allosteric communication over several nanometers, and suggest that disorder in the dimeric arrangement of the effector domains is important for phosphatase activity in a two-component system. The results have implications for the development of optogenetic applications.Peer reviewe