1.E.1 The P21 Holin S (P21 Holin) Family Phage 21 S holin is the prototype for class II holins. It has two TMSs with both the N- and C-termini on the cytoplasmic side of the inner membrane of E. coli (Gründling et al. 2000). As for other holins, it functions in the export of an endolysin, but the holin channel also allows release of small ions and metabolites (Park et al. 2006). TMS1 may be dispensable for function. A homologue of the P21 holin is the holin of bacteriophage H-19B. The gene encoding it has been associated with the Shiga-like Toxin I gene in E. coli (Neely and Friedman, 1998). It may function in toxin export as has been proposed for the X. nematophila holin-1 (Brillard et al., 2003; TC #1.E.2.1.4). These and other cell lysis systems have been reviewed (Cahill and Young 2019).
This family belongs to the Holin II Superfamily .
References:
Ahammad, T., D.L. Drew, Jr, I.D. Sahu, R.A. Serafin, K.R. Clowes, and G.A. Lorigan. (2019). Continuous Wave Electron Paramagnetic Resonance Spectroscopy Reveals the Structural Topology and Dynamic Properties of Active Pinholin S68 in a Lipid Bilayer. J Phys Chem B 123: 8048-8056.
Ahammad, T., R.H. Khan, I.D. Sahu, D.L. Drew, Jr, E. Faul, T. Li, R.M. McCarrick, and G.A. Lorigan. (2021). Pinholin S mutations induce structural topology and conformational changes. Biochim. Biophys. Acta. Biomembr 1863: 183771.
Astashkin, R., K. Kovalev, S. Bukhdruker, S. Vaganova, A. Kuzmin, A. Alekseev, T. Balandin, D. Zabelskii, I. Gushchin, A. Royant, D. Volkov, G. Bourenkov, E. Koonin, M. Engelhard, E. Bamberg, and V. Gordeliy. (2022). Structural insights into light-driven anion pumping in cyanobacteria. Nat Commun 13: 6460.
Brillard, J., M.-H. Boyer-Giglio, N. Boemare, and A. Givaudan. (2003). Holin locus characterisation from lysogenic Xenorhabdus nematophila and its involvement in Escherichia coli SheA haemolytic phenotype. FEMS Microbiol. Lett. 218: 107-113.
Cahill, J. and R. Young. (2019). Phage Lysis: Multiple Genes for Multiple Barriers. Adv Virus Res 103: 33-70.
Drew, D.L., Jr, T. Ahammad, R.A. Serafin, I.D. Sahu, R.H. Khan, E. Faul, R.M. McCarrick, and G.A. Lorigan. (2021). Probing the local secondary structure of bacteriophage S pinholin membrane protein using electron spin echo envelope modulation spectroscopy. Biochim. Biophys. Acta. Biomembr 1864: 183836. [Epub: Ahead of Print]
Govorunova, E.G., O.A. Sineshchekov, R. Hemmati, R. Janz, O. Morelle, M. Melkonian, G.K. Wong, and J.L. Spudich. (2018). Extending the Time Domain of Neuron.al Silencing with Cryptophyte Anion Channelrhodopsins. eNeuro 5:.
Gründling, A., U. Bläsi, and R. Young. (2000). Biochemical and genetic evidence for three transmembrane domains in the class I holin, lambda S. J. Biol. Chem. 275: 769-776.
Khan, R.H., T. Ahammad, I.D. Sahu, N.C. Rotich, A. Daufel, and G.A. Lorigan. (2023). Determining the helical tilt angle and dynamic properties of the transmembrane domains of pinholin S68 using mechanical alignment EPR spectroscopy. Biochim. Biophys. Acta. Biomembr 1865: 184154.
Neely, M.N. and D.I. Friedman. (1998). Functional and genetic analysis of regulatory regions of coliphage H-19B: location of shiga-like toxin and lysis genes suggest a role for phage functions in toxin release. Mol. Microbiol. 28: 1255-1267.
Pang, T., C.G. Savva, K.G. Fleming, D.K. Struck, and R. Young. (2009). Structure of the lethal phage pinhole. Proc. Natl. Acad. Sci. USA 106: 18966-18971.
Park, T., D.K. Struck, C.A. Dankenbring, and R. Young. (2007). The pinholin of lambdoid phage 21: control of lysis by membrane depolarization. J. Bacteriol. 189(24):9135-9139.
Park, T., D.K. Struck, J.F. Deaton, and R. Young. (2006). Topological dynamics of holins in programmed bacterial lysis. Proc. Natl. Acad. Sci. USA 103: 19713-19718.
Pasqua, M., A. Zennaro, R. Trirocco, G. Fanelli, G. Micheli, M. Grossi, B. Colonna, and G. Prosseda. (2021). Modulation of OMV Production by the Lysis Module of the DLP12 Defective Prophage of. Microorganisms 9:.
Srividhya, K.V. and S. Krishnaswamy. (2007). Subclassification and targeted characterization of prophage-encoded two-component cell lysis cassette. J Biosci 32: 979-990.
Steger, L.M.E., A. Kohlmeyer, P. Wadhwani, J. Bürck, E. Strandberg, J. Reichert, S.L. Grage, S. Afonin, M. Kempfer, A.C. Görner, J. Koch, T.H. Walther, and A.S. Ulrich. (2020). Structural and functional characterization of the pore-forming domain of pinholin S68. Proc. Natl. Acad. Sci. USA 117: 29637-29646.
Watari, M., T. Ikuta, D. Yamada, W. Shihoya, K. Yoshida, S.P. Tsunoda, O. Nureki, and H. Kandori. (2019). Spectroscopic study of the transmembrane domain of a rhodopsin-phosphodiesterase fusion protein from a unicellular eukaryote. J. Biol. Chem. 294: 3432-3443.
Examples:
TC#	Name	Organismal Type	Example
Examples:
TC#	Name	Organismal Type	Example
1.E.1.1.1	Lysis protein S; also called ''pinholin'' or pinholin S(21)68, of 71 aas and 2 TMSs. It forms small heptameric pores that depolarize the membrane (Park et al., 2007; Pang et al., 2009). This holin is of topological Class II, forming 2 TMSs, with the N- and C-termini inside (Park et al. 2006). TMS1 partially externalizes from the lipid bilayer regulates channel-formation and interacts with the membrane surface, whereas TMS2 remains buried in the lipid bilayer in the active conformation and forms the pore (Ahammad et al. 2019). Pinholin S(21)68 triggers the lytic cycle of bacteriophage phi21 in infected Escherichia coli. Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Structural models have been proposed for (1) the oligomeric pinhole (right-handed heptameric TMD2 bundle), (2) the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and (3) the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line) (Steger et al. 2020). The TMSs are α-helical rather than pi or 310-helices which have been observed in other channel forming proteins (Drew et al. 2021). Pinholin S(21) mutations induce structural, topological and conformational changes (Ahammad et al. 2021). The helical tilt angle and dynamic properties of the transmembrane domains of pinholin S²¹68 have been determined using mechanical alignment EPR spectroscopy (Khan et al. 2023).	E. coli, phage P21	Lysis protein S (71 aas; P27360)

1.E.1.1.2	Lysis protein S. Identical to EssD, a holin from lambdoid prophage DLP12 with two TMSs (Srividhya and Krishnaswamy 2007).	*E. coli*	Lysis protein S (71aas; P77237)

1.E.1.1.3	Holin of 68 aas and 1 TMS. Deleting the lysis module, encoding the holin, lysin and two spanins, increases outer membrane vesicle (OMV) production, suggesting that during evolution this operon has been domesticated to regulate vesiculation, possibly through the elimination of non-recyclable peptidoglycan fragments (Pasqua et al. 2021). The expression of the lysis module is negatively regulated by environmental stress stimuli as high osmolarity, low pH and low temperature (Pasqua et al. 2021).	E. coli phage H-19B	*Holin of E. coli* phage H-19B (68 aas; AAD04658)**

1.E.1.1.4	Lysis S family holin protein	Bacteria	Lysis S holin of E. coli

1.E.1.1.5	Hypothetical protein, HP	Bacteria	HP of Cronobacter sakazakii

1.E.1.1.6	Holin of 62 aas and 1 TMS	γ-Proteobacteria	Putative holin of Hamiltonella defensa

1.E.1.1.7	Lysis S family protein with fused N-terminal holin domain of 2 - 3 TMSs; 720 aas	Proteobacteria	Holin-Lysis S fusion protein of E. coli

1.E.1.1.8	Holin of 86 aas and 1 TMS.		Holin of Pectobacterium polonicum

1.E.1.1.9	Putative uncharacterized holin of 75 aas and 1 or 2 TMSs. Shows extensive similarity to members of TC family 1.E.25.		Holin of Salmonella enterica

Examples:
TC#	Name	Organismal Type	Example
Examples:
TC#	Name	Organismal Type	Example
Examples:
TC#	Name	Organismal Type	Example