Structural Basis for the Recognition and Cleavage of Polysialic Acid by the Bacteriophage K1F Tailspike Protein EndoNF Eike Christian Schulz 1 , David Schwarzer 2 , Martin Frank 3 , Katharina Stummeyer 2 , Martina Mühlenhoff 2 , Achim Dickmanns 1 , Rita Gerardy-Schahn 2 ⁎ and Ralf Ficner 1 ⁎ 1 Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Justus-von-Liebig- Weg 11, 37077 Göttingen, Germany 2 Institut für Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg- Strasse 1, 30625 Hannover, Germany 3 Deutsches Krebsforschungszentrum, Molekulare Strukturanalyse, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Received 11 November 2009; received in revised form 11 January 2010; accepted 13 January 2010 Available online 22 January 2010 An α-2,8-linked polysialic acid (polySia) capsule confers immune tolerance to neuroinvasive, pathogenic prokaryotes such as Escherichia coli K1 and Neisseria meningitidis and supports host infection by means of molecular mimicry. Bacteriophages of the K1 family, infecting E. coli K1, specifically recognize and degrade this polySia capsule utilizing tailspike endosiali- dases. While the crystal structure for the catalytic domain of the endosialidase of bacteriophage K1F (endoNF) has been solved, there is yet no structural information on the mode of polySia binding and cleavage available. The crystal structure of activity deficient active-site mutants of the homotrimeric endoNF cocrystallized with oligomeric sialic acid identified three independent polySia binding sites in each endoNF monomer. The bound oligomeric sialic acid displays distinct conformations at each site. In the active site, a Sia 3 molecule is bound in an extended conformation representing the enzyme–product complex. Structural and biochemical data supported by molecular modeling enable to propose a reaction mechanism for polySia cleavage by endoNF. © 2010 Elsevier Ltd. All rights reserved. Edited by G. Schulz Keywords: polysialic acid; endoNF; glycosidase; host recognition Introduction On the surface of mammalian cells, polysialic acid (poly-α2,8-linked Neu5Ac; polySia) plays an impor- tant role in cellular motility and neuronal plasticity, and has implications in tumor metastasis. 1 This natural abundance triggers tolerance of the immune system to polySia. 2 Thus, a thick polySia capsule expressed by neuroinvasive bacteria such as Escher- ichia coli K1 and Neisseria meningitidis serogroup B conveys resistance to the host immune system and serves as a disguise mechanism during host invasion. 3 However, in order to gain access to the bacterial surface and to inject their genetic material, bacteriophages of the K1 family evolved the ability to specifically recognize and degrade the bacterial polySia capsule utilizing tailspike proteins with endosialidase activity. 4–10 In contrast to exosiali- dases (E.C. 3.2.1.18), which remove single, terminal sialic acid moieties from sialoglycoconjugates, endo- sialidases (E.C. 3.2.1.129) are highly specific for polySia and cleave within the polymer chain. *Corresponding authors. E-mail addresses: Gerardy-Schahn.Rita@mh-hannover.de; rficner@gwdg.de. Abbreviations used: polySia, polysialic acid; PDB, Protein Data Bank; TBA, thiobarbituric acid; MD, molecular dynamics; TMB, 3,3′,5,5′-tetramethyl- benzidine; BSA, bovine serum albumin; PBS, phosphate- buffered saline. doi:10.1016/j.jmb.2010.01.028 J. Mol. Biol. (2010) 397, 341–351 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.