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• W2888820744 abstract "In all herpesviruses, the space between the capsid shell and the lipid envelope is occupied by the unique tegument layer composed of proteins that, in addition to structural roles, play many other roles in the viral replication. UL37 is a highly conserved tegument protein that has activities ranging from virion morphogenesis to directional capsid trafficking to manipulation of the host innate immune response and binds multiple partners. The N-terminal half of UL37 (UL37N) has a compact bean-shaped α-helical structure that contains a surface region essential for neuroinvasion. However, no biochemical or structural information is currently available for the C-terminal half of UL37 (UL37C) that mediates most of its interactions with multiple binding partners. Here, we show that the C-terminal half of UL37 from pseudorabies virus UL37C is a conformationally flexible monomer composed of an elongated folded core and an unstructured C-terminal tail. This elongated structure, along with that of its binding partner UL36, explains the nature of filamentous tegument structures bridging the capsid and the envelope. We propose that the dynamic nature of UL37 underlies its ability to perform diverse roles during viral replication. In all herpesviruses, the space between the capsid shell and the lipid envelope is occupied by the unique tegument layer composed of proteins that, in addition to structural roles, play many other roles in the viral replication. UL37 is a highly conserved tegument protein that has activities ranging from virion morphogenesis to directional capsid trafficking to manipulation of the host innate immune response and binds multiple partners. The N-terminal half of UL37 (UL37N) has a compact bean-shaped α-helical structure that contains a surface region essential for neuroinvasion. However, no biochemical or structural information is currently available for the C-terminal half of UL37 (UL37C) that mediates most of its interactions with multiple binding partners. Here, we show that the C-terminal half of UL37 from pseudorabies virus UL37C is a conformationally flexible monomer composed of an elongated folded core and an unstructured C-terminal tail. This elongated structure, along with that of its binding partner UL36, explains the nature of filamentous tegument structures bridging the capsid and the envelope. We propose that the dynamic nature of UL37 underlies its ability to perform diverse roles during viral replication. Herpesviridae are a large family of viruses that infect a broad range of hosts from mollusks to birds to humans. All herpesviruses are composed of a capsid containing the dsDNA genome surrounded by a layer of proteins called the tegument, which is enclosed by the glycoprotein-studded lipid envelope. The tegument layer is stabilized by extensive protein/protein interactions and is largely structurally maintained even once the viral membrane is removed (1.McLauchlan J. Rixon F.J. Characterization of enveloped tegument structures (L particles) produced by alphaherpesviruses: integrity of the tegument does not depend on the presence of capsid or envelope.J. Gen. Virol. 1992; 73 (1311356): 269-27610.1099/0022-1317-73-2-269Crossref PubMed Scopus (99) Google Scholar). The tegument includes 23 distinct virally encoded proteins, in addition to several host proteins (2.Owen D.J. Crump C.M. Graham S.C. Tegument assembly and secondary envelopment of alphaherpesviruses.Viruses. 2015; 7 (26393641): 5084-511410.3390/v7092861Crossref PubMed Scopus (121) Google Scholar). The tegument is further subdivided into the inner and outer tegument. The inner tegument is directly associated with the capsid, and protein copy numbers are tightly controlled. The outer tegument is less ordered and associates with the viral envelope instead of the capsid (2.Owen D.J. Crump C.M. Graham S.C. Tegument assembly and secondary envelopment of alphaherpesviruses.Viruses. 2015; 7 (26393641): 5084-511410.3390/v7092861Crossref PubMed Scopus (121) Google Scholar). In addition to linking the capsid to the lipid envelope (3.Jambunathan N. Chouljenko D. Desai P. Charles A.S. Subramanian R. Chouljenko V.N. Kousoulas K.G. Herpes simplex virus 1 protein UL37 interacts with viral glycoprotein gK and membrane protein UL20 and functions in cytoplasmic virion envelopment.J. Virol. 2014; 88 (24600000): 5927-593510.1128/JVI.00278-14Crossref PubMed Scopus (37) Google Scholar), tegument proteins play additional roles during viral infection (2.Owen D.J. Crump C.M. Graham S.C. Tegument assembly and secondary envelopment of alphaherpesviruses.Viruses. 2015; 7 (26393641): 5084-511410.3390/v7092861Crossref PubMed Scopus (121) Google Scholar). The inner tegument protein UL36, at ∼330 kDa, the largest tegument protein (4.Kelly B.J. Fraefel C. Cunningham A.L. Diefenbach R.J. Functional roles of the tegument proteins of herpes simplex virus type 1.Virus Res. 2009; 145 (19615419): 173-18610.1016/j.virusres.2009.07.007Crossref PubMed Scopus (106) Google Scholar), is involved in capsid transport (5.Luxton G.W. Lee J.I. Haverlock-Moyns S. Schober J.M. Smith G.A. The pseudorabies virus VP1/2 tegument protein is required for intracellular capsid transport.J. Virol. 2006; 80 (16352544): 201-20910.1128/JVI.80.1.201-209.2006Crossref PubMed Scopus (110) Google Scholar, 6.Schipke J. Pohlmann A. Diestel R. Binz A. Rudolph K. Nagel C.H. Bauerfeind R. Sodeik B. The C terminus of the large tegument protein pUL36 contains multiple capsid binding sites that function differently during assembly and cell entry of herpes simplex virus.J. Virol. 2012; 86 (22258258): 3682-370010.1128/JVI.06432-11Crossref PubMed Scopus (68) Google Scholar), release of viral DNA (7.Abaitua F. Hollinshead M. Bolstad M. Crump C.M. O'Hare P. A nuclear localization signal in herpesvirus protein VP1–2 is essential for infection via capsid routing to the nuclear pore.J. Virol. 2012; 86 (22718835): 8998-901410.1128/JVI.01209-12Crossref PubMed Scopus (56) Google Scholar), and virion maturation (6.Schipke J. Pohlmann A. Diestel R. Binz A. Rudolph K. Nagel C.H. Bauerfeind R. Sodeik B. The C terminus of the large tegument protein pUL36 contains multiple capsid binding sites that function differently during assembly and cell entry of herpes simplex virus.J. Virol. 2012; 86 (22258258): 3682-370010.1128/JVI.06432-11Crossref PubMed Scopus (68) Google Scholar, 8.Fuchs W. Klupp B.G. Granzow H. Mettenleiter T.C. Essential function of the pseudorabies virus UL36 gene product is independent of its interaction with the UL37 protein.J. Virol. 2004; 78 (15479829): 11879-1188910.1128/JVI.78.21.11879-11889.2004Crossref PubMed Scopus (101) Google Scholar, 9.Desai P.J. A null mutation in the UL36 gene of herpes simplex virus type 1 results in accumulation of unenveloped DNA-filled capsids in the cytoplasm of infected cells.J. Virol. 2000; 74 (11090159): 11608-1161810.1128/JVI.74.24.11608-11618.2000Crossref PubMed Scopus (181) Google Scholar) and contains a deubiquitinating domain (10.Kattenhorn L.M. Korbel G.A. Kessler B.M. Spooner E. Ploegh H.L. A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae.Mol. Cell. 2005; 19 (16109378): 547-55710.1016/j.molcel.2005.07.003Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Other tegument proteins are involved in regulation of viral and host gene expression. For example, the outer tegument protein UL48, also known as α-transactivator, activates transcription of immediate-early viral genes (11.Campbell M.E. Palfreyman J.W. Preston C.M. Identification of herpes simplex virus DNA sequences which encode a trans-acting polypeptide responsible for stimulation of immediate early transcription.J. Mol. Biol. 1984; 180 (6096556): 1-1910.1016/0022-2836(84)90427-3Crossref PubMed Scopus (332) Google Scholar). Another outer tegument protein, UL41, is an endonuclease that suppresses host protein expression by degrading mRNA and is commonly termed vhs (viral host shutoff) (12.Smiley J.R. Herpes simplex virus virion host shutoff protein: immune evasion mediated by a viral RNase?.J. Virol. 2004; 78 (14722261): 1063-106810.1128/JVI.78.3.1063-1068.2004Crossref PubMed Scopus (176) Google Scholar). Likewise, the inner tegument protein UL37 plays multiple roles in the viral replication cycle. Along with UL36 (13.Leege T. Granzow H. Fuchs W. Klupp B.G. Mettenleiter T.C. Phenotypic similarities and differences between UL37-deleted pseudorabies virus and herpes simplex virus type 1.J. Gen. Virol. 2009; 90 (19297610): 1560-156810.1099/vir.0.010322-0Crossref PubMed Scopus (36) Google Scholar, 14.Vittone V. Diefenbach E. Triffett D. Douglas M.W. Cunningham A.L. Diefenbach R.J. Determination of interactions between tegument proteins of herpes simplex virus type 1.J. Virol. 2005; 79 (16014918): 9566-957110.1128/JVI.79.15.9566-9571.2005Crossref PubMed Scopus (170) Google Scholar, 15.Klupp B.G. Fuchs W. Granzow H. Nixdorf R. Mettenleiter T.C. Pseudorabies virus UL36 tegument protein physically interacts with the UL37 protein.J. Virol. 2002; 76 (11861875): 3065-307110.1128/JVI.76.6.3065-3071.2002Crossref PubMed Scopus (133) Google Scholar), which is directly bound to the viral capsid (16.Dai X. Zhou Z.H. Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes.Science. 2018; 360 (29622628): eaao729810.1126/science.aao7298Crossref PubMed Scopus (98) Google Scholar), UL37 initiates tegument accumulation on the capsid during virion assembly and forms filamentous structures that may serve as a scaffold for the outer tegument proteins (17.Grünewald K. Desai P. Winkler D.C. Heymann J.B. Belnap D.M. Baumeister W. Steven A.C. Three-dimensional structure of herpes simplex virus from cryo-electron tomography.Science. 2003; 302 (14631040): 1396-139810.1126/science.1090284Crossref PubMed Scopus (449) Google Scholar, 18.Laine R.F. Albecka A. van de Linde S. Rees E.J. Crump C.M. Kaminski C.F. Structural analysis of herpes simplex virus by optical super-resolution imaging.Nat. Commun. 2015; 6 (25609143): 598010.1038/ncomms6980Crossref PubMed Scopus (94) Google Scholar). In HSV-1, 3The abbreviations used are: HSV-1herpes simplex virus-1PRVpseudorabies virusUL37CC terminus of UL37UL37NN terminus of UL37SECsize-exclusion chromatographyMALSmulti-angle light scatteringSAXSsmall angle X-ray scatteringRgradius of gyrationDmaxmaximum dimensionEOMensemble optimization modelingBS3bis(sulfosuccinimidyl)suberateSUMOsmall ubiquitin-like modifierC-tailC-terminal tailGSTglutathione S-transferaseLBlysogeny brothTCEPtris(2-carboxyethyl)phosphineCAPS3-(cyclohexylamino)propanesulfonic acidPDBProtein Data BankNSDnormalized spatial discrepancy. UL37 promotes formation of the enveloped viral particles, termed secondary envelopment, by binding viral envelope proteins gK and UL20, thereby linking capsid and envelope (3.Jambunathan N. Chouljenko D. Desai P. Charles A.S. Subramanian R. Chouljenko V.N. Kousoulas K.G. Herpes simplex virus 1 protein UL37 interacts with viral glycoprotein gK and membrane protein UL20 and functions in cytoplasmic virion envelopment.J. Virol. 2014; 88 (24600000): 5927-593510.1128/JVI.00278-14Crossref PubMed Scopus (37) Google Scholar). Beyond structure and assembly, UL37 homologs from HSV-1 and PRV have been shown to facilitate efficient capsid trafficking during both viral entry and egress. During entry into epithelial cells, HSV-1 and PRV viruses lacking UL37 are delayed in trafficking, with about 50% of capsids failing to reach the nucleus (13.Leege T. Granzow H. Fuchs W. Klupp B.G. Mettenleiter T.C. Phenotypic similarities and differences between UL37-deleted pseudorabies virus and herpes simplex virus type 1.J. Gen. Virol. 2009; 90 (19297610): 1560-156810.1099/vir.0.010322-0Crossref PubMed Scopus (36) Google Scholar). During egress, in the absence of UL37, HSV-1 and PRV capsids do not efficiently traffic from the nucleus to the sites of secondary envelopment (vesicles derived from Golgi (19.Turcotte S. Letellier J. Lippé R. Herpes simplex virus type 1 capsids transit by the trans-Golgi network, where viral glycoproteins accumulate independently of capsid egress.J. Virol. 2005; 79 (15994778): 8847-886010.1128/JVI.79.14.8847-8860.2005Crossref PubMed Scopus (125) Google Scholar) or endosomes (20.Hollinshead M. Johns H.L. Sayers C.L. Gonzalez-Lopez C. Smith G.L. Elliott G. Endocytic tubules regulated by Rab GTPases 5 and 11 are used for envelopment of herpes simplex virus.EMBO J. 2012; 31 (22990238): 4204-422010.1038/emboj.2012.262Crossref PubMed Scopus (99) Google Scholar)), and “naked” capsids accumulate within the cytoplasm (21.Desai P. Sexton G.L. McCaffery J.M. Person S. A null mutation in the gene encoding the herpes simplex virus type 1 UL37 polypeptide abrogates virus maturation.J. Virol. 2001; 75 (11581394): 10259-1027110.1128/JVI.75.21.10259-10271.2001Crossref PubMed Scopus (94) Google Scholar, 22.Klupp B.G. Granzow H. Mundt E. Mettenleiter T.C. Pseudorabies virus UL37 gene product is involved in secondary envelopment.J. Virol. 2001; 75 (11533156): 8927-893610.1128/JVI.75.19.8927-8936.2001Crossref PubMed Scopus (97) Google Scholar). In HSV-1, UL37 has been shown to promote capsid transport on microtubules during egress in epithelial cells through its interaction with host trafficking protein dystonin/BPAG1a (23.Pasdeloup D. McElwee M. Beilstein F. Labetoulle M. Rixon F.J. Herpesvirus tegument protein pUL37 interacts with dystonin/BPAG1 to promote capsid transport on microtubules during egress.J. Virol. 2013; 87 (23269794): 2857-286710.1128/JVI.02676-12Crossref PubMed Scopus (53) Google Scholar). UL37 plays an even more prominent role in capsid trafficking in neurons during infection with HSV-1 and PRV. UL37-null viruses are defective in retrograde movement from axon termini to the nucleus and avirulent in the mouse model of infection, demonstrating the importance of efficient capsid trafficking in viral replication (24.Richards A.L. Sollars P.J. Pitts J.D. Stults A.M. Heldwein E.E. Pickard G.E. Smith G.A. The pUL37 tegument protein guides α-herpesvirus retrograde axonal transport to promote neuroinvasion.PLoS Pathog. 2017; 13 (29216315): e100674110.1371/journal.ppat.1006741Crossref PubMed Scopus (49) Google Scholar). Finally, HSV-1 UL37 manipulates the innate immune response by binding immune modulators TNF receptor–associated factor 6 (TRAF6) (25.Liu X. Fitzgerald K. Kurt-Jones E. Finberg R. Knipe D.M. Herpesvirus tegument protein activates NF-κB signaling through the TRAF6 adaptor protein.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18682563): 11335-1133910.1073/pnas.0801617105Crossref PubMed Scopus (75) Google Scholar) and retinoic acid-inducible gene I (RIG-I) (26.Zhao J. Zeng Y. Xu S. Chen J. Shen G. Yu C. Knipe D. Yuan W. Peng J. Xu W. Zhang C. Xia Z. Feng P. A viral deamidase targets the helicase domain of RIG-I to block RNA-induced activation.Cell Host Microbe. 2016; 20 (27866900): 770-78410.1016/j.chom.2016.10.011Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Binding of UL37 to TRAF6 leads to activation of transcription factor NF-κB, which subsequently localizes to the nucleus and turns on expression of immune response genes, such as interleukin-8 (IL-8), presumably to increase transcription of early viral genes (25.Liu X. Fitzgerald K. Kurt-Jones E. Finberg R. Knipe D.M. Herpesvirus tegument protein activates NF-κB signaling through the TRAF6 adaptor protein.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18682563): 11335-1133910.1073/pnas.0801617105Crossref PubMed Scopus (75) Google Scholar). UL37 also deamidates RIG-I, thus preventing its activation by viral dsRNA, which decreases the overall interferon production and stunts the antiviral response (26.Zhao J. Zeng Y. Xu S. Chen J. Shen G. Yu C. Knipe D. Yuan W. Peng J. Xu W. Zhang C. Xia Z. Feng P. A viral deamidase targets the helicase domain of RIG-I to block RNA-induced activation.Cell Host Microbe. 2016; 20 (27866900): 770-78410.1016/j.chom.2016.10.011Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). herpes simplex virus-1 pseudorabies virus C terminus of UL37 N terminus of UL37 size-exclusion chromatography multi-angle light scattering small angle X-ray scattering radius of gyration maximum dimension ensemble optimization modeling bis(sulfosuccinimidyl)suberate small ubiquitin-like modifier C-terminal tail glutathione S-transferase lysogeny broth tris(2-carboxyethyl)phosphine 3-(cyclohexylamino)propanesulfonic acid Protein Data Bank normalized spatial discrepancy. The structural characteristics underlying multiple distinct activities of UL37 are unknown. We previously reported the crystal structures of the N-terminal half of UL37 (UL37N) from PRV and HSV-1 (27.Pitts J.D. Klabis J. Richards A.L. Smith G.A. Heldwein E.E. Crystal structure of the herpesvirus inner tegument protein UL37 supports its essential role in control of viral trafficking.J. Virol. 2014; 88 (24599989): 5462-547310.1128/JVI.00163-14Crossref PubMed Scopus (32) Google Scholar, 28.Koenigsberg A.L. Heldwein E.E. Crystal structure of the N-terminal half of the traffic controller UL37 from herpes simplex virus 1.J. Virol. 2017; 91 (28768862): e01244-1710.1128/JVI.01244-17Crossref PubMed Scopus (11) Google Scholar) that revealed the bean-shaped structure of UL37N and helped identify a conserved surface region essential for directional capsid trafficking during neuroinvasion (24.Richards A.L. Sollars P.J. Pitts J.D. Stults A.M. Heldwein E.E. Pickard G.E. Smith G.A. The pUL37 tegument protein guides α-herpesvirus retrograde axonal transport to promote neuroinvasion.PLoS Pathog. 2017; 13 (29216315): e100674110.1371/journal.ppat.1006741Crossref PubMed Scopus (49) Google Scholar). However, no structural information is yet available for the C-terminal domain of UL37 (UL37C) that mediates the majority of known interactions of UL37. Here, we show that UL37C forms an elongated, conformationally flexible species with a folded core and an unstructured C-tail. We hypothesize that, in addition to providing a large surface with many potential binding sites for its multiple binding partners, UL37C can adopt several distinct conformations, each of which mediates a specific subset of UL37 activities. We propose that this conformational flexibility contributes to the multifunctionality of UL37. During expression in Escherichia coli, most of PRV UL37FL is cleaved, which generates the stable N-terminal half but no detectable C-terminal fragment (27.Pitts J.D. Klabis J. Richards A.L. Smith G.A. Heldwein E.E. Crystal structure of the herpesvirus inner tegument protein UL37 supports its essential role in control of viral trafficking.J. Virol. 2014; 88 (24599989): 5462-547310.1128/JVI.00163-14Crossref PubMed Scopus (32) Google Scholar) (Fig. 1A). Affinity tagging both the N and C termini of UL37FL allowed for its purification but did not improve the yield. Therefore, the following constructs of PRV UL37C were cloned, expressed, and purified instead: UL37C(478–919)-StII, UL37C(478–884), UL37C(499–884), and UL37C(618–919)-StII (Fig. 1B). Although the last resolved residue of the crystal structure of PRV UL37N is Leu-479, only Trp-477 was unambiguously assigned to UL37N due to extensive interactions within the core. Therefore, conservatively, UL37C starts at residue 478, and UL37C(478–919)-StII represents the largest UL37C construct. UL37C(478–884) lacks a large portion of the unstructured C-terminal tail, whereas UL37C(499–884) additionally lacks the unstructured N-terminal region (Fig. S1). Finally, UL37C(618–919)-StII was designed based on a stable UL37 degradation product observed during expression of HSV-1 UL37 in mammalian cells. 4P. Feng, unpublished observations. Upon size-exclusion chromatography (SEC), all PRV UL37C constructs migrated as apparent dimers or trimers (Fig. 2A). However, SEC-coupled multi-angle light scattering (SEC-MALS) estimated molecular masses of PRV UL37C(478–919)-StII and UL37C(478–884) to be 50 and 46 kDa, which is close to their respective theoretical molecular masses of 48 and 43 kDa. Thus, PRV UL37C is a monomer in solution (Fig. 2, B and C). To rule out concentration-dependent oligomerization, SEC-MALS of UL37C(478–884) was performed at a range of concentrations: 1, 3, and 5 mg/ml. At all three concentrations, UL37C(478–884) was a monomer with an estimated molecular mass of 46 kDa (data not shown). To confirm the lack of dimerization, the PRV UL37C constructs were incubated with bis(sulfosuccinimidyl)suberate (BS3), a homobifunctional cross-linking reagent that reacts with primary amines, including the N-terminal amine and lysine side chains. Monomeric PRV UL37N (27.Pitts J.D. Klabis J. Richards A.L. Smith G.A. Heldwein E.E. Crystal structure of the herpesvirus inner tegument protein UL37 supports its essential role in control of viral trafficking.J. Virol. 2014; 88 (24599989): 5462-547310.1128/JVI.00163-14Crossref PubMed Scopus (32) Google Scholar) was used as a negative control. No high-molecular-mass species were observed for any construct in the presence of the cross-linker, consistent with a lack of oligomerization (Fig. 2D). The largest UL37C construct, UL37C(478–919)-StII, migrated at ∼55 kDa on SDS-PAGE (Fig. 2D), higher than its theoretical molecular mass of 48 kDa, yet migrated at ∼48 kDa after incubation with BS3. By contrast, UL37C(478–884) migrated at ∼43 kDa with or without cross-linker (Fig. 2D). The increased mobility of UL37C(478–919)-StII is indicative of intramolecular cross-linking, which prevents complete unfolding under SDS-PAGE conditions. An apparent lack of intramolecular cross-linking in UL37C(478–884), which lacks the second half of the unstructured C-terminal tail, suggests that BS3 reacts with a lysine side chain near the C terminus. UL37C has high α-helical content, according to its CD spectra (Fig. 2E), but is predicted to have an unstructured C-terminal tail (Fig. S1). We used limited proteolysis to map the boundaries of the folded core of UL37C. Digests of UL37C(478–919)-StII with either chymotrypsin or trypsin resulted in a single major proteolytic product migrating at ∼40 kDa, with the trypsin-generated product being slightly larger (Fig. 3, A and B). N-terminal sequencing of both 40-kDa proteolytic products showed that both start with the GPGS sequence, generated by cleavage of the N-terminal His6-SUMO tag by PreScission protease during protein purification (Fig. 1B), implying that trypsin and chymotrypsin cleavage leave the N terminus intact. Neither degradation product retained the C-terminal StII tag, as determined by streptavidin–horseradish peroxidase Western blotting (data not shown). Thus, both proteases cleave UL37C near the C terminus. Mass spectrometry analysis of the chymotrypsin digest showed a predominant peak at 41.8 kDa, consistent with cleavage after residue Leu-866 (Fig. 3C and Fig. S2) (theoretical mass of 41.6 kDa). Trypsin generated a product ∼2 kDa larger, which is consistent with cleavage after residue Arg-884 (theoretical mass 43.5 kDa) (Fig. 3C). According to the secondary structure prediction, both proteolytic sites are located within the unstructured C-terminal tail (Fig. 3C). To assess the overall stability of UL37C, we used the Thermofluor, or differential scanning fluorimetry, assay (29.Phillips, K., and de la Pena, A. H. (2011) The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization Curr. Protoc. Mol. Biol, Chapter 10, Unit 10.28 10.1002/0471142727.mb1028s94 21472694Google Scholar), which estimates the melting temperature (Tm) by measuring binding of a hydrophobic dye to hydrophobic regions exposed during unfolding. Thermal melting was done in 24 different buffer and salt conditions to identify those that could potentially stabilize the protein. The highest Tm for UL37C(478–919)-StII and UL37C(478–884) was 49 and 45 °C, respectively (Fig. 1C, Fig. S3, and Table S1). By contrast, the highest Tm for PRV UL37N is 61 °C (Fig. 1C, Fig. S3, and Table S1). The highest Tm for any construct fell within a pH range of 7.0–7.5 and increased with the addition of 150 mm NaCl and 5–10% glycerol (Table S1). Regardless of buffer conditions, the lowest Tm for PRV UL37N was still higher than any Tm for PRV UL37C. The relatively low melting temperature of UL37C could be due to a weak hydrophobic core (30.Franzosa E.A. Lynagh K.J. Xia Y. Structural correlates of protein melting temperature.in: Experimental Standard Conditions of Enzyme Characterizations. Beilstein-Institut, Ruedesheim/Rhein, Germany2009: 99-106Google Scholar). We have been unable to crystallize any PRV UL37C constructs despite extensive efforts. To obtain structural information on UL37C, we employed small-angle X-ray scattering coupled with SEC (SEC-SAXS), which is used to characterize conformational state and flexibility of macromolecules in solution. SAXS profiles were obtained for three constructs of PRV UL37C and for HSV-1 UL37N, a compact, conformationally rigid protein that served as a control (Fig. 4 and Fig. 5A). PRV UL37C(478–919)-StII and HSV-1 UL37N had only one intensity peak, whereas UL37C(478–884) and UL37C(618–919)-StII each had two peaks. The later peaks correspond to a smaller radius of gyration (Rg) around 5–10 Å, potentially due to degradation products (Fig. 4, B and C). The Rg profile across the primary peak for all samples slopes downward, but frames chosen for analyses had Rg values within ±1 Å of each other. Parameters calculated from SAXS data are summarized in Table 1.Figure 5SAXS analysis of PRV UL37C and HSV-1 UL37N. A, scattering profiles are shown after averaging and subtraction of solvent scattering from solid and dashed boxes, respectively, in Fig. 4. I(q) versus q are shown as log-linear plots. B, Guinier plots (including linear fits) at the low-angle region (qRg < 1.3). Data points in gray were excluded from the Guinier region. C, distance distribution functions shown for when forced to zero (inset) and not forced to zero (primary panel). Faded lines, curve after the determined Dmax. D, dimensionless Kratky plots were calculated from scattering curves in A. Data for PRV UL37C(478–919)-StII and HSV-1 UL37N have been deposited into the SASBDB under codes SASDDQ9 and SASDDP9, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1SAXS structural parametersUL37C(478–919)-StIIUL37C(478–884)HSV-1 UL37NGuinier analysis I(0) (cm−1)aI(0) = scattering intensity at zero angle.0.017 ± 0.000070.0081 ± 0.000070.02786 ± 0.00008 Rg (Å)bRadius of gyration.41.97 ± 0.9538.86 ± 0.5133.34 ± 0.14 qmin (Å−1)cqmin = lowest q value used in the Guinier analysis.0.008550.01080.00912 qmaxRgdMaximum q value used in the Guinier analysis times Rg.1.292081.307191.29265 Coefficient of correlation, R2eLinear fit of the Guinier plot.0.99090.96950.9968P(r) analysis (forced to 0/not forced)fP(r) forced to 0 at Dmax in DATGNOM/P(r) not forced to 0. I(0) (cm−1)0.017 ± 0.00008/0.019 ± 0.00040.0078 ± 0.00004/0.0085 ± 0.00020.02785 ± 0.00005 Rg (Å)43.00 ± 0.17/50.2 ± 0.3039.34 ± 0.16/47.63 ± 0.3833.81 ± 0.065 Dmax (Å)gMaximum dimension calculated from P(r).140/250125/250120 q range (Å−1)hq values used in P(r) plot.0.007–0.2808/0.007–0.28080.007–0.2808/0.007–0.28080.007–0.2808 χ2 (total estimate from GNOM)iχ2 fit of P(r) to data.,jTotal quality estimate from GNOM.1.169 (0.80)/1.087 (0.68)1.112 (0.80)/1.055 (0.66)1.106 (0.88)DAMAVER Symmetry,kSymmetry used for DAMMIN input. anisotropy assumptionslProlate, oblate, or none for DAMMIN input.P1, noneP1, noneP1, none Models averagedmNumber of DAMMIN models averaged using DAMAVER. (omitted)nNumber of DAMMIN models omitted from DAMAVER if NSD > mean + 2 × S.D.10 (0)10 (0)10 (0) Mean NSDoMean NSD for each model used in DAMAVER average. (S.D.)0.619 (0.014)0.660 (0.017)0.644 (0.014) χ2 rangepRange of χ2 values for input DAMMIN models.1.186–1.1971.172–1.1751.013–1.015 ResolutionqResolution of final filtered average from DAMAVER. (from SASRES (48.Tuukkanen A.T. Kleywegt G.J. Svergun D.I. Resolution of ab initio shapes determined from small-angle scattering.IUCrJ. 2016; 3 (27840683): 440-44710.1107/S2052252516016018Crossref PubMed Scopus (61) Google Scholar)) (Å)41 ± 342 ± 338 ± 3a I(0) = scattering intensity at zero angle.b Radius of gyration.c qmin = lowest q value used in the Guinier analysis.d Maximum q value used in the Guinier analysis times Rg.e Linear fit of the Guinier plot.f P(r) forced to 0 at Dmax in DATGNOM/P(r) not forced to 0.g Maximum dimension calculated from P(r).h q values used in P(r) plot.i χ2 fit of P(r) to data.j Total quality estimate from GNOM.k Symmetry used for DAMMIN input.l Prolate, oblate, or none for DAMMIN input.m Number of DAMMIN models averaged using DAMAVER.n Number of DAMMIN models omitted from DAMAVER if NSD > mean + 2 × S.D.o Mean NSD for each model used in DAMAVER average.p Range of χ2 values for input DAMMIN models.q Resolution of final filtered average from DAMAVER. Open table in a new tab For PRV UL37C(478–919)-StII, UL37C(478–884), and HSV-1 UL37N, SAXS-derived molecular masses (31.Rambo R.P. Tainer J.A. Accurate assessment of mass, models and resolution by small-angle scattering.Nature. 2013; 496 (23619693): 477-48110.1038/nature12070Crossref PubMed Scopus (526) Google Scholar) were consistent with the theoretical values (Fig. 1C), and Guinier plots were linear, which indicated good data quality and no significant protein aggregation (Fig. 5B). By contrast, the SAXS-derived molecular mass of PRV UL37C(618–919)-StII was 85 kDa, which is more than twic" @default.
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• W2888820744 date "2018-10-01" @default.
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• W2888820744 title "The dynamic nature of the conserved tegument protein UL37 of herpesviruses" @default.
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