title: An intramolecular interaction involving the N terminus of a streptococcal adhesin affects its conformation and adhesive function.
abstract: BACKGROUND
  P1 is an adhesin on the surface of Streptococcus mutans.
  
  
  RESULTS
  Destroying the high affinity interaction between the N and C termini of S. mutans P1 creates a non-adherent phenotype.
  
  
  CONCLUSIONS
  The N terminus facilitates proper folding, function, and stability within recombinant P1.
  
  
  CONCLUSIONS
  The relationship between folding, maturation, and cell surface assembly is critical to understanding the P1 mechanism of action. The adhesin P1 is localized on the surface of the oral pathogen Streptococcus mutans and facilitates an interaction with the glycoprotein complex salivary agglutinin that is comprised primarily of the scavenger receptor gp340. Recent crystal structures of P1 display an unusual structure in which the protein folds back upon itself to form an elongated hybrid helical stalk with a globular head at the apex and a globular C-terminal region at the base. The N terminus of P1 has not yet been characterized. In this report we describe the contribution of an interaction between the N-terminal and C-terminal portions of the protein that is required for proper function of P1 on the surface of S. mutans. Utilizing recombinant N-terminal and C-terminal fragments, we employed isothermal titration calorimetry and native gel electrophoresis to demonstrate that these fragments form a high affinity and stable complex in solution. Furthermore, circular dichroism and surface plasmon resonance measurements indicated that the N-terminal fragment contributes to the folding and increases the functionality of the C-terminal fragment in trans. Finally, we utilized circular dichroism, surface plasmon resonance, and differential scanning calorimetry to show that an N-terminal 106-amino acid segment within P1 contributes to the proper folding and function of the full-length recombinant molecule and increases the stability of its elongated hybrid helical stalk.
authors.affiliation: Department of Oral Biology, University of Florida, Gainesville, Florida 32610, USA.
authors.forename: Kyle P
authors.initials: KP
authors.lastname: Heim
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Crowley
authors.forename: L Jeannine
authors.initials: LJ
authors.lastname: Brady
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000373:Agglutinins / Q000737:chemistry / Q000378:metabolism; D001422:Bacterial Adhesion; D002942:Circular Dichroism; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D058849:Protein Refolding; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013295:Streptococcus mutans / Q000378:metabolism* / Q000502:physiology; D013816:Thermodynamics
chemical_list: D018829:Adhesins, Bacterial; D000373:Agglutinins; D055767:Immobilized Proteins; D010446:Peptide Fragments
keywords: Bacterial Adhesion; Cell Surface Protein; Circular Dichroism (CD); Isothermal Titration Calorimetry; Protein Conformation; Protein Folding; Streptococcus; Surface Plasmon Resonance (SPR)
references.citation: Mol Microbiol. 2005 Mar;55(5):1591-605
references.pmid: 15720563
references.citation: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Jan 1;67(Pt 1):23-6
references.pmid: 21206016
references.citation: Adv Exp Med Biol. 1992;327:229-41
references.pmid: 1295342
references.citation: Infect Immun. 1990 Feb;58(2):289-96
references.pmid: 2298480
references.citation: Infect Immun. 1991 Dec;59(12):4425-35
references.pmid: 1937801
references.citation: Infect Immun. 2004 Jan;72(1):13-21
references.pmid: 14688075
references.citation: Infect Immun. 1980 May;28(2):486-93
references.pmid: 6995311
references.citation: J Gen Microbiol. 1979 Sep;114(1):109-15
references.pmid: 118231
references.citation: J Biol Chem. 2004 Nov 12;279(46):47699-703
references.pmid: 15355985
references.citation: Infect Immun. 2005 Apr;73(4):2245-52
references.pmid: 15784568
references.citation: J Biol Chem. 2009 Jul 31;284(31):20729-37
references.pmid: 19497855
references.citation: Minerva Stomatol. 1990 May;39(5):413-29
references.pmid: 2199814
references.citation: J Am Chem Soc. 2011 Jan 26;133(3):478-85
references.pmid: 21142157
references.citation: Protein Sci. 1999 Feb;8(2):370-80
references.pmid: 10048330
references.citation: Microbiology (Reading). 2012 Dec;158(Pt 12):2903-2916
references.pmid: 23082034
references.citation: Infect Immun. 1989 Dec;57(12):3702-7
references.pmid: 2807544
references.citation: Innate Immun. 2010 Jun;16(3):160-7
references.pmid: 20418254
references.citation: Arch Oral Biol. 1990;35 Suppl:33S-38S
references.pmid: 1982405
references.citation: Biochemistry. 1992 Apr 28;31(16):3947-55
references.pmid: 1567847
references.citation: Methods Mol Biol. 2009;572:101-33
references.pmid: 20694688
references.citation: J Biol Chem. 2002 Aug 30;277(35):32109-15
references.pmid: 12050164
references.citation: Science. 1994 Feb 11;263(5148):777-84
references.pmid: 8303294
references.citation: Eur J Biochem. 1983 Jun 15;133(2):255-61
references.pmid: 6852037
references.citation: Infect Immun. 2003 Feb;71(2):676-81
references.pmid: 12540545
references.citation: Infect Immun. 1998 Sep;66(9):4274-82
references.pmid: 9712778
references.citation: J Immunol. 2009 Oct 1;183(7):4628-38
references.pmid: 19752237
references.citation: J Biol Chem. 2011 Jun 17;286(24):21657-66
references.pmid: 21505225
references.citation: Oral Microbiol Immunol. 1989 Jun;4(2):106-11
references.pmid: 2762013
references.citation: J Biol Chem. 2004 May 21;279(21):22198-203
references.pmid: 15060072
references.citation: Methods Mol Biol. 2005;305:1-16
references.pmid: 15939991
references.citation: Microbiol Rev. 1980 Jun;44(2):331-84
references.pmid: 6446023
references.citation: Curr Protoc Cell Biol. 2004 Sep;Chapter 17:Unit 17.8
references.pmid: 18228446
references.citation: Adv Protein Chem. 1992;43:313-61
references.pmid: 1442323
references.citation: Methods Enzymol. 2004;383:318-51
references.pmid: 15063656
references.citation: Crit Rev Biochem Mol Biol. 2001;36(5):399-433
references.pmid: 11724155
references.citation: Aust Dent J. 2000 Dec;45(4):235-45
references.pmid: 11225524
references.citation: Protein Eng. 2000 Sep;13(9):617-27
references.pmid: 11054456
references.citation: Microbiol Mol Biol Rev. 2009 Sep;73(3):407-50, Table of Contents
references.pmid: 19721085
references.citation: Infect Immun. 1992 Mar;60(3):1008-17
references.pmid: 1541515
references.citation: Infect Immun. 1998 Jan;66(1):115-21
references.pmid: 9423847
references.citation: Infect Immun. 2004 Aug;72(8):4680-8
references.pmid: 15271929
references.citation: Infect Immun. 1989 Mar;57(3):805-9
references.pmid: 2521840
references.citation: G Batteriol Virol Immunol. 1990 Jan-Dec;83(1-12):108-17
references.pmid: 2133318
references.citation: Infect Immun. 2008 Jun;76(6):2456-68
references.pmid: 18362133
references.citation: J Gen Microbiol. 1986 Oct;132(10):2873-83
references.pmid: 2957461
references.citation: J Mol Biol. 2010 Apr 2;397(3):740-51
references.pmid: 20138058
references.citation: J Biol Chem. 2010 Oct 29;285(44):33858-66
references.pmid: 20729215
references.citation: FEMS Immunol Med Microbiol. 2007 Aug;50(3):342-53
references.pmid: 17535300
references.citation: J Sch Nurs. 2005 Oct;21(5):272-8
references.pmid: 16262438
references.citation: Arch Oral Biol. 1990;35 Suppl:15S-23S
references.pmid: 2088219
references.citation: J Bacteriol. 1995 Sep;177(17):5028-34
references.pmid: 7665480
references.citation: J Mol Biol. 1992 Sep 5;227(1):293-306
references.pmid: 1522594
references.citation: Infect Immun. 1987 Nov;55(11):2759-67
references.pmid: 3312011
references.citation: Infect Immun. 1993 Nov;61(11):4590-8
references.pmid: 7691754
references.citation: J Dent Res. 1994 Sep;73(9):1493-502
references.pmid: 7523469
references.citation: Infect Immun. 1999 Mar;67(3):1201-6
references.pmid: 10024561
references.citation: Biochemistry. 1981 Mar 3;20(5):1085-94
references.pmid: 7225319
references.citation: Infect Immun. 1989 Nov;57(11):3306-13
references.pmid: 2807526
references.citation: Infect Immun. 1993 Apr;61(4):1547-52
references.pmid: 8454362
references.citation: Infect Immun. 2004 Aug;72(8):4699-706
references.pmid: 15271931
references.citation: Methods Enzymol. 1986;130:208-69
references.pmid: 3773734
references.citation: Proc Natl Acad Sci U S A. 2010 Mar 30;107(13):5983-8
references.pmid: 20231452
references.citation: J Microbiol Methods. 2006 Jun;65(3):503-11
references.pmid: 16239043
references.citation: Anal Biochem. 2000 Dec 15;287(2):252-60
references.pmid: 11112271
references.citation: Biochemistry. 1991 Apr 30;30(17):4237-44
references.pmid: 2021617
references.citation: Proteins. 1995 Aug;22(4):404-12
references.pmid: 7479713
references.citation: Anal Biochem. 1986 May 15;155(1):155-67
references.pmid: 3717552
references.citation: Methods Mol Biol. 2002;173:121-6
references.pmid: 11859755
references.citation: Infect Immun. 1992 Dec;60(12):5057-64
references.pmid: 1333448
references.citation: Proc Natl Acad Sci U S A. 2009 Oct 6;106(40):16967-71
references.pmid: 19805181
references.citation: Infect Immun. 1991 May;59(5):1803-10
references.pmid: 1673448
references.citation: Infect Immun. 1991 Dec;59(12):4606-9
references.pmid: 1937820
references.citation: Biol Chem. 2008 Sep;389(9):1193-200
references.pmid: 18713006
references.citation: J Mol Biol. 1995 Sep 22;252(3):337-50
references.pmid: 7563055