Minicollagen-15, a Novel Minicollagen Isolated from Hydra, Forms Tubule Structures in Nematocysts Patrizia Adamczyk 1 , Sebastian Meier 2 , Thomas Gross 1 , Bert Hobmayer 3 , Stephan Grzesiek 4 , Hans Peter Bächinger 5 , Thomas W. Holstein 1 ⁎ and Suat Özbek 1 ⁎ 1 Institute of Zoology, Department of Molecular Evolution and Genomics, Im Neuenheimer Feld 230, 69130 Heidelberg, Germany 2 Institute of Molecular Biology and Physiology, August Krogh Building, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark 3 Institute of Zoology and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria 4 Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland 5 Shriners Hospital for Children and Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA Received 23 May 2007; received in revised form 26 October 2007; accepted 31 October 2007 Available online 5 December 2007 Minicollagens constitute a family of unusually short collagen molecules isolated from cnidarians. They are restricted to the nematocyst, a cylindrical explosive organelle serving in defense and capture of prey. The nematocyst capsule contains a long tubule inside of its matrix, which is expelled and everted during an ultrafast discharge process. Here, we report the cloning and characterization of a novel minicollagen in Hydra, designated mini- collagen-15 (NCol-15). NCol-15, like NCol-3 and NCol-4, shows deviations from the canonical cysteine pattern in its terminal cysteine-rich domains (CRDs). Minicollagens share common domain architectures with a central collagen sequence flanked by polyproline stretches and short N- and C- terminal CRDs. The CRDs are involved in the formation of a highly resistant cysteine network, which constitutes the basic structure of the nematocyst capsule. Unlike NCol-1, which is part of the capsule wall, NCol-15 is localized to tubules, arguing for a functional differentiation of minicollagens within the nematocyst architecture. NMR analysis of the altered C-terminal CRD of NCol-15 showed a novel disulfide-linked structure within the cysteine-containing region exhibiting similar folding kinetics and stability as the canonical CRDs. Our data provide evidence for evolutionary diver- sification among minicollagens, which probably facilitated alterations in the morphology of the nematocyst wall and tubule. © 2007 Elsevier Ltd. All rights reserved. Edited by M. F. Summers Keywords: minicollagen; Hydra; nematocyst; evolution; cysteine-rich domain *Corresponding authors. E-mail addresses: holstein@uni-hd.de; soezbek@zoo.uni-heidelberg.de. Abbreviations used: CRD, cysteine-rich domain; BrdU, bromodeoxyuridine; ROESY, rotating frame Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser enhancement; GSH, reduced glutathione; GSSG, oxidized glutathione; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RDC, residual dipolar coupling. doi:10.1016/j.jmb.2007.10.090 J. Mol. Biol. (2008) 376, 1008–1020 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.