The X-Ray Crystal Structure of the Phage λ Tail Terminator Protein Reveals the Biologically Relevant Hexameric Ring Structure and Demonstrates a Conserved Mechanism of Tail Termination among Diverse Long-Tailed Phages

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The X-Ray Crystal Structure of the Phage λ Tail Terminator Protein Reveals the Biologically Relevant Hexameric Ring Structure and Demonstrates a Conserved Mechanism of Tail Termination among Diverse Long-Tailed Phages

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  The X-Ray Crystal Structure of the Phage  λ  TailTerminator Protein Reveals the Biologically RelevantHexameric Ring Structure and Demonstrates aConserved Mechanism of Tail Termination amongDiverse Long-Tailed Phages Lisa G.Pell 1,2 , Amanda Liu 3 , Lizbeth Edmonds 4 , Logan W. Donaldson 4 ,P. Lynne Howell 1,2 and Alan R. Davidson 1,3 ⁎ 1 Department of Biochemistry,Faculty of Medicine, Universityof Toronto, Medical SciencesBuilding, Toronto, ON, Canada, M5S 1A8 2  Molecular Structure andFunction, Research Institute, The Hospital for Sick Children, 555University Avenue, Toronto,ON, Canada, M5G 1X8 3 Department of MolecularGenetics, University of Toronto, Medical Sciences Building,Toronto, ON, Canada, M5S 1A8 4 Department of Biology, York University, 4700 Keele Street,Toronto, ON, Canada, M3J 1P3Received 27 January 2009;received in revised form23 April 2009;accepted 28 April 2009 Available online6 May 2009The tail terminator protein (TrP) plays an essential role in phage tailassembly by capping the rapidly polymerizing tail once it has reached itsrequisite length and serving as the interaction surface for phage heads.Here, we present the 2.7-Å crystal structure of a hexameric ring of gpU, theTrP of phage  λ . Using sequence alignment analysis and site-directedmutagenesis, we have shown that this multimeric structure is biologicallyrelevant and we have delineated its functional surfaces. Comparison of thehexameric crystal structure with the solution structure of gpU that wepreviously solved using NMR spectroscopy shows large structural changesoccurringuponmultimerizationandsuggestsamechanismthatallowsgpUto remain monomeric at high concentrations on its own, yet polymerizereadily upon contact with an assembled tail tube. The gpU hexamerdisplays several flexible loops that play key roles in head and tail binding,implying a role for disorder-to-order transitions in controlling assembly ashas been observed with other  λ  morphogenetic proteins. Finally, we havefound that the hexameric structure of gpU is very similar to the structure of a putative TrP from a contractile phage tail even though it displays nodetectable sequence similarity. This finding coupled with further bioinfor-matic investigations has led us to conclude that the TrPs of non-contractile-tailed phages, such as  λ , are evolutionarily related to those of contractile-tailed phages, such as P2 and Mu, and that all long-tailed phages mayutilize a conserved mechanism for tail termination. © 2009 Elsevier Ltd. All rights reserved. Edited by R. Huber  Keywords:  bacteriophage  λ ; tail assembly; tail termination; crystal structure;evolution Introduction The majority of bacteriophages possess icosahe-dral double-stranded-DNA-filled heads and long( ∼ 50 – 200 nm) tails, 1 which are crucial for host cellrecognition, cell wall penetration, and viral genomedelivery. These long-tailed phages can be dividedinto two distinct groups: those with contractile tails(  Myoviridae ) and those with non-contractile tails( Siphoviridae ). 2 In the contractile-tailed phages, atail sheath protein surrounds the tail tube andcontracts upon infection, allowing the tail tube topenetrate the host cell. In contrast, non-contractiletails lack a sheath and do not change shapesignificantly upon infection. Since phage tailscomprise a complex arrangement of multiple copiesof many different proteins that must interact in a *Corresponding author.  E-mail address:alan.davidson@utoronto.ca.Abbreviations used: TrP, tail terminator protein; TMP,tape measure protein; PDB, Protein Data Bank; WT, wildtype; ORF,open reading frame; Se-Met, selenomethionine. doi:10.1016/j.jmb.2009.04.072  J. Mol. Biol.  (2009)  389 , 938 – 951  Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.  precisely ordered pathway, elucidation of themechanisms by which tails assemble has presenteda fascinating challenge.One issue pertaining to tail assembly that hasattracted considerable attention over the years is theregulation of length. 3,4 Within any particular speciesofphage,virtuallyeveryparticlepossessesatailwithprecisely the same length. The exact length of bothcontractile and non-contractile tails is set by a tapemeasure protein (TMP) where the tail length isproportional to the length of the TMP. 5 – 7 However,an additional protein, known as the tail terminatorprotein (TrP), is required to prevent aberrant poly-merization of the tail tube protein beyond the lengthencoded by the TMP (Fig. 1). The product of the bacteriophage  λ  U   gene (gpU) is the best character-ized TrP. In the absence of gpU, λ infections result inthe formation of normal heads, which are producedin a pathway separate from tail assembly, andextremely elongated tails, which result from theuncontrolled polymerization of the tail tube proteingpV. In the completed tail structure, gpU assemblesat the top of the tail tube, 8 thereby terminating gpVpolymerization and providing the surface for inter-action with the head. 9 gpU is presumed to constitutea hexameric ring within the tail structure because itspontaneously forms such rings in the presence of Mg 2+ that match the size of the hexameric rings of gpV that comprise the bulk of the tail tube. 8 However, prior to tail assembly, gpU remainsmonomeric even at millimolar concentrations,which allowed us to solve the solution structure of this form of the protein using NMR spectroscopy. 10 Since all long-tailed phages must avoid uncon-trolled tail tube polymerization, it is expected thatthey all encode a protein with tail terminatingactivity. Consistent with this notion, mutations thatled to elongated tails similar to those observed incells infected by phage  λ  U   mutants have beenidentified in the contractile-tailed phages Mu, P2,T4, and SPO1. 8,10 – 14 However, the question of whetherallofthesephagesusethesamemechanismfor tail termination remains open because theirputative TrPs have not been characterized and nosequence similarity can be detected among them.An indication that the structure of TrPs may beconserved in diverse phages was provided by astrong similarity detected between the structure of gpU and the structure of protein STM4215 [ProteinData Bank (PDB) ID: 2GJV] encoded in a prophage element of   Salmonella typhimurium . 10 Although thisprophage element is uncharacterized, the geneencoding STM4215 lies adjacent to other genesencoding components of a contractile tail, suggest-ing that it may be a TrP. Interestingly, STM4215crystallized as a hexameric ring with dimensionssimilar to the ring of gpV seen in the  λ  tail, whichstrengthens the notion that it is indeed a TrP.Nevertheless, without knowledge of the biologi-cally relevant quaternary structure of a  bona fide TrP, it is impossible to arrive at definitive conclu-sions pertaining to the function of STM4215 and thepotential for conservation of TrP structure andfunction among diverse phages.In the current work, we have pursued furtherstudies on phage  λ  gpU. Despite its relatively smallsize (14.8 kDa), this protein presents an intriguingobject for investigation, possessing surfaces tomediate self-interaction and binding to both thephage head and tail. In addition, gpU multimerizesupon contact with the appropriate tail assemblyintermediate, yet remains monomeric at high proteinconcentrations on its own. Knowledge of its biolo-gically relevant quaternary structure is essential tofully understand the mechanisms by which gpUperforms its functions. As is described here, we havesolved a hexameric structure of gpU using X-raycrystallography. Although the formation of thisstructure was induced by crystallization, we haveemployed bioinformatic and mutagenesis studiesto show that this structure is biologically relevantand have also identified the functional surfaces of gpU. Using this structure as a starting point, weperformed further bioinformatic investigation totrace the evolutionary relationships between gpUand diverse TrPs from contractile-tailed phages. Thiswork complements recently published work fromour laboratory showing that the tail tube proteins of contractile andnon-contractilephagesare likelytobeevolutionarily related. 15 Fig. 1.  Bacteriophage  λ  tail assembly. (a) In the WTpathway, an initiator complex is formed (green) followed by the polymerization of gpV (blue) on top of theinitiator, a process that is regulated by the TMP, gpH(orange). Once the precise tail length is reached, gpVpolymerization is terminated by gpU (purple) at pre-cisely 32 gpV hexamers. The tail is then activated by gpZ,and an assembled  λ  head binds to gpU, forming aninfectious phage particle. (b) In the absence of gpU, gpVpauses briefly at the correct tail length and thencontinues to polymerize, forming extremely long struc-tures called polytails. (c) If gpU is present but contains amutation to its top surface, it will successfully bind to  λ tails, terminating gpV polymerization at the correct taillength, but will be incapable of interacting with  λ  heads.(d) Conversely, if gpU is present but contains a mutationto its bottom surface, tail termination will not occur and apolytail phenotype, similar to those produced in a gpU − phage, is observed. 939 The Structure of the   λ  Tail Terminator Protein   Results The X-ray crystal structure of gpU Even though we had already determined thetertiary structure of gpU using NMR spectroscopy, 10 we initiated crystallization studies on this proteinwith the hope that the conditions of crystallizationwould induce formation of a biologically relevantmultimer as has been observed in other systems. 16,17 Since diffraction-quality crystals of gpU wild type(gpU-WT) were not obtained initially, we also testedmutants that had been investigated previously. 10 Fortunately,diffraction-qualitycrystalswereobtainedwith one of these mutants, gpU-D74A, in severaldifferent conditions containing ammonium sulfate.Ultimately, the structure of this protein was solved to2.7 Å using selenomethionyl incorporation and thesingle-wavelengthanomalousdispersionmethodandrefined to an  R cryst  and  R free  of 0.233 and 0.283,respectively (Table 1).As we had hoped, the asymmetric unit of gpU-D74A contained two almost identical hexamericrings.Theseringswere84Åindiameterwitha30-Å-diameter central pore (Fig. 2a), a size that is veryclose to the dimensions of the hexameric rings of gpV that comprise the tail tube, 20 suggesting thatthey might be biologically relevant. Subsequent tosolving the gpU-D74A structure, crystals of gpU-WT were grown and the structure was solved to2.8 Å using molecular replacement (Table 1). ThegpU-WT (PDB ID: 3FZB) and gpU-D74A (PDB ID:3FZ2) monomeric structures were very similar withan overall RMSD of 0.55 Å  21 over aligned C α atoms between chain A and chain C, respectively. Surpris-ingly, however, the asymmetric unit of the gpU-WTcrystalscontainedtwopentamericringsofgpUwitha diameter of 74 Å and a 27-Å central pore (Fig. 2 b).Despite this difference in stoichiometry, the inter-subunit interfaces seen in the gpU-WT and gpU-D74A structures were almost identical (Fig. 2c). It Table 1.  Diffraction and refinement statistics gpU-D74A gpU-WT Data collection statistics Space group  P 2 1 2 1 2 1  P 12 1 1Cell dimensions a  (Å) 87.37 74.10 b  (Å) 125.40 127.99 c  (Å) 211.61 82.91 α  (°) 90 90 β  (°) 90 108.92 γ  (°) 90 90Wavelength (Å ) 0.9800 1.5418Resolution (Å) a 46.84 – 2.70(2.80 – 2.70)70.10 – 2.80(2.90 – 2.80)No. of reflections 563,982 141,896No. of unique reflections 123,413 36,086Redundancy a 4.57 (4.57) 3.93 (3.93)Completeness (%) a 99.9 (100) 99.9 (100) R merge b (%) a 0.083 (0.289) 0.099 (0.312)Average  I  / σ ( I  ) a 8.8 (3.7) 8.6 (3.4) Refinement statisticsR cryst / R free  (%) c 23.3/28.3 23.6/30.9No. of protein atoms 12,258 9748No. of water molecules 246 109RMSD from ideal geometryBond lengths (Å) 0.007 0.008Angles (°) 1.4 1.5Estimated coordinateerror (Å) d 0.49 0.68Average  B -factor (Å  2 ) 42.55 47.39Ramachandran plot (%) e 86.6/13.2/0.1/0 82.6/16.8/0.6/0 a Values in parentheses are for the outer resolution shell,2.80 – 2.70 (gpU-D74A) and 2.90 – 2.80 (gpU-WT). b R merge  = P hkl P N  j  = 1 j I  hkl    I  hkl  j ð Þj = P hkl N  4 I  hkl , where  I  hkl  is thediffraction intensity of an individual measurement over  N  data sets. c R cryst  = P hkl jj F obs j   k  j F calc jj = P hkl j F obs j , where  F obs  and  F calc  arethe observed and calculated structure factors, respectively, and  k  is the scale factor. d Obtained from CNS-SOLVE. 18 e DeterminedwithPROCHECK. 19 (Percentageofresiduesfoundin the most favored/additionally allowed/generously allowed/disallowed regions of the plot.) Fig. 2.  The quaternary structure of gpU. (a) The gpU-D74A hexamer and (b) gpU-WT pentamer are shown incartoon representation. The subunits of each oligomer arecolored in varying shades of blue. (c) Ribbon representa-tion of the superposition of a dimer from the gpU-WT(yellow) asymmetric unit with a dimer from the gpU-D74A (blue) asymmetric unit. Several residues buried inthe inter-subunit interface in each structure are repre-sented as sticks. (d) The asymmetric unit of gpU-D74A (atleft) and gpU-WT (at right) contains two hexameric andpentameric rings, respectively. The subunits from the firsthexamer/pentamer are colored in varying shades of blue,while the subunits from the second hexamer/pentamerare colored in varying shades of green. 940  The Structure of the   λ  Tail Terminator Protein   should be noted that in both the gpU-D74A and WTstructures, the two rings found in the asymmetricunit are arranged face-to-face across a 2-fold non-crystallographic axis and an approximately 2-foldnon-crystallographic axis with a translation, respec-tively (Fig. 2d). We do not believe that thisarrangement is biologically relevant because itwould produce a closed structure that would notallow the gpU ring to interact with two completelydifferent surfaces (i.e., the head and the tail).The interaction of two gpU molecules in thegpU-D74A hexamer buries 1165 Å  2 of its mono-meric surface area, 18 a degree of burial that is quitetypical for biologically relevant protein – proteininteractions. 22 Eleven hydrophobic/aromatic andeight charged/polar amino acid side chains aresignificantly buried by hexamer formation as shownin Fig. 3a. A potentially important network of electrostatic and hydrogen bond interactions occur between the side chains from Ser75, Asp78, andTyr109 from one monomer and His3, Thr4, Arg7,and Asp25 from an adjacent monomer (Fig. 3 b). Thehexameric structure may also be stabilized by aninter-subunit antiparallel  β -sheet formed between ashort segment of   β 2 (residues 46 – 48) and a segmentof   β 4 (residues 105 – 107) in a second monomer. Anintermolecular backbone H-bond also occurs between positions 25 and 74. As can be seen inFig. 2c, the orientation of the side chains buried inthe inter-subunit interface are almost identical in theWT and gpU-D74A structures.Superposition of the 12 molecules of gpU in theasymmetric unit revealed minimal structural devia-tions (backbone RMSD, 0.77±0.22 Å) in the regionspossessing regular secondary-structure elements;however, three loop regions spanning residues 28to 36 ( β 1 – β 2 loop), 50 to 57 ( β 2 – β 3 loop), and 111 to117 ( β 4 – β 5 loop) did not overlay well (Fig. 4a).Poorly defined electron density and higher-than-averagetemperaturefactorswerealsonotedintheseregions of the model. The moderate resolution andsomewhat high  R cryst  and  R free  observed for thisstructuremightbeattributedtothepresenceoftheseflexible loops. The monomeric structure of gpU seenin both the WT and D74A crystal forms possesses Fig. 3.  The oligomerization interface of gpU. (a)Nineteen residues are found buried in the inter-subunitinterface of the gpU-D74A hexamer. Interface residuesthat are at least 80% conserved among gpU homologuesare colored red while residues that exhibit less than 80%conservation are colored yellow. (b) An electrostatic andhydrogen bond network is shown between residues His3,Thr4, Arg7, and Asp25 from one monomer (colored blue)and between Ser75, Asp78, and Tyr109 from a secondmonomer (colored green). Residues participating incontacts at the interface were determined using CNS 18 and PISA. 23 Fig. 4.  Comparison of the NMR and X-ray structure of gpU. (a) The superposition of the 12 asymmetric unitmonomers of gpU-D74A (at left) with the  β 1 – β 2,  β 2 – β 3,and  β 4 – β 5 loops circled in orange. On the right, thecartoon representation of a gpU monomer from the X-raystructure allows for easy visualization of the mixed  α / β -fold. The sheet ( β 1,  β 2,  β 3,  β 5, and  β 4), primarilycomposed of antiparallel strands with the exception of  β 1, which runs parallel with  β 2, is flanked on one surface by three  α -helices and one 3 10 -helix. Secondary-structureelements are labeled on the gpU-D74A structure wherestrands are in dark blue, the helices are in cyan, and loopsare in gray. The two structural representations are related by ∼ 180°.(b)Theloop-de-loopmotifofagpUmonomerishighlighted in red. (c) Overlay of the lowest-energy NMRstructure (red) with one chain from the X-ray crystalstructure (blue). Thr100 is highlighted with spheres ineach structure. The  β 2 – β 3 loop is colored green in the X-ray structure and yellow in the NMR structure.(d)Secondary-structure comparison between the NMR (red)and X-ray (blue) structures as defined by DSSP. 24 Theloop-de-loop motif is boxed. In (a) – (c), the top and bottomare noted with respect to how each molecule is orientedwith respect to a hexamer. Structures should not becompared between panels as they are each shown indifferent orientations. 941 The Structure of the   λ  Tail Terminator Protein   the same mixed α - and β -fold as we observed in ourprevious NMR study. 10 This fold consists of a four-stranded antiparallel  β -sheet packed on one sideagainst three  α -helices (Fig. 4a). Both the gpU X-rayand NMR structures also feature an unusual  “ loop-de-loop ”  motif (residues 22 – 37) joining  α 1 and  β 2where the short  β 1 strand (residues 22 – 25) is joinedto  β 2 with a circular loop resulting in a parallelinteraction between  β 1 and  β 2 (Fig. 4 b). In spite of  their overall similarity, significant deviations wereobserved between the NMR and X-ray structuressuch that the optimal overlay of all residues in thetwo structures yields a very high backbone atomRMSD of 5.4 Å (Fig. 4c). Interestingly, almost all of the major deviations between these structures occurin the bottom quarter (as oriented in Fig. 4c) witheach of the secondary-structure elements in thisregion ( α 1 and  β 2 – β 5) being shortened by betweenthree and five residues in the NMR structure(Fig. 4d). The most extreme structural deviationsare seen just past the end of   α 3 where 15 Å separates the Thr100 position in the two structuresand in the loop between  β 2 and  β 3 where the NMRstructure displays a large (residues 43 – 60), poorlyconstrained loop, while the X-ray structure pos-sesses a much tighter turn in this region spanningfar fewer residues (residues 49 – 56) (Fig. 4c and d).The differences between the NMR and X-raystructures are unlikely to be due simply to one of these structures being poorly determined sincegreater than 60% of their residues can be overlaidwell to produce a backbone atom RMSD of only1.9 Å, and the large structural changes are localized(Fig. 4c). Interestingly, the regions of greatestdeviation are involved in the formation of theinter-subunit  β -sheet interaction mentioned above.It may be the oligomerization process and forma-tion of these inter-subunit H-bonds that induce thestructural differences seen between the monomericNMR structure and the oligomeric crystal structure. Conservation analysis of an alignment ofgpU homologues As a means to verify the biological relevance of our crystal structure and to identify key residues ingpU, we constructed an alignment of gpU homo-logues from a variety of prophages. The sequencesin this alignment were selected for maximal diver-sity from a much larger collection of  sequencesidentified through an iterated PSI-BLAST 25 search.Due to the increase in sequenced bacterial genomes,we were able to construct a much more diverse andinformative alignment than was shown in ourprevious work 10 such that the average pairwiseidentity between sequences in this alignment was25%, and no pair of sequences was more than 45%identical. A strong correlation between the con-served features in this alignment and our structureofgpU can bedetected.Forexample,more than80%of the29hydrophobiccore positions in our structuredisplayed high conservation of hydrophobic resi-dues (Fig. 5). Importantly, greater than 50% of the 19positions occupied by residues significantly buriedin the inter-subunit interface showed strong con-servation. In contrast, conservation was seen at only14% of the other surface-exposed positions of gpU.The high degree of conservation of residues lying atthe subunit interface provides strong support for the biological relevance of the hexameric structure of gpU. A smaller number of conserved positions werefound on the top and bottom surfaces of the ring,and another conserved group of residues forms a buriedclusterincludingresiduesintheloop-de-loopregion on the inner surface of the gpU ring (Fig. 5).Strikingly, no conserved residues were found on the Fig. 5.  Alignment of gpU homologues. The alignment was generated as described in Materials and Methods. Theaverage pairwise identity of these sequences is 25% with no pair of sequences being more than 45% identical. Yellow andred circles above the alignment denote residues found in the hydrophobic core and the oligomerization interface,respectively. Additionally, several residues on the top (T) and bottom (B) surface of the gpU hexamer are noted. Positionsthat show conservation in 80% of the sequences are highlighted. Hydrophobic residues are colored light blue, aromaticresidues are colored green, positive residues are colored red, and negative residues are colored magenta. Aromaticresidues at predominantly hydrophobic positions were considered to be conserved. 942  The Structure of the   λ  Tail Terminator Protein 
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