讲稿分子医学

1、FtsZ Protofilaments Use a Hinge Opening Mechanism for Constrictive Force GenerationSheng Ye, Ph.D.Life Sciences InstituteZhejiang UniversityFtsZ: A molecular machano-chemical device nascent septum (S)FtsZ forms a ring associated with divisionBi & Lutkenhaus, Nature 1991FtsZ dynamics during the divis

2、ion cycleMa & Margolin, PNAS 1996FtsZ: A molecular machano-chemical device FtsZ itself can assemble the Z ring and generate a forceOsawa et al, Science 2008FtsZ is a distinct family of GTPasesFtsZ topological map and 3-D structureThe FtsZ N-terminal domain binds GTPGTP binding siteGTP + H2O GDP+ PO4

3、FtsZTwo FtsZ monomers assemble in a head-to-tail manner forming a complete GTPaseQuestion:How does FtsZ ring convert the chemical energy of GTP hydrolysis to a mechanical constrictive force?Table : X-ray data and refinement statisticsData setSpace GroupUnit CellResolution ()Measured reflectionsUniqu

4、e reflectionsRedundancyCompleteness (%, highest shell)Mean I/I (highest shell)Rsym (%, highest shell)P6422a=132.5 , c=321.5 2.9790,24737,41421.199.8 (100.0)65.7 (2.2)13.4 (63.4)RefinementResolution ()No. of reflections F0 FR-factor/R-free (%)No. of Protein AtomsNo. of GDP moleculesNo. of Water Molec

5、ulesrmsd bond lengths ()rmsd bond angles ()Ramachandran PlotMost favoured regions (%)Additional allowed regions (%)Generously allowed regions (%)Disallowed regions (%)2.935,81524.1/26.76,371300.0061.095.34.700Question #1Whether the observed inter-subunit interface is biologically relevant or is just

6、 a crystallographic artifact?The hydrophobic residues involved in the inter-subunit interface are highly conserved.In vivo characterization of Escherichia coli ftsZ MutantsA complementation system = an ftsZ knock-out strain + a temperature-sensitive rescue plasmid + a complementation plasmid control

7、led by the pBAD promoterFtsZRegular E. Coli30 CftsZ null E. Coli (JKD7-1 strain)30 CJKD7-1 strain + pKD3 vectorFtsZ30 CFtsZpKD3 rescue plasmidconstantly expresses FtsZFtsZJKD7-1 strain + pKD3 vector42 CFtsZpKD3 plasmid is temperature-sensitive for replicationFtsZFtsZJKD7-1 strain + pKD3 vector + pJS

8、B vector42 CFtsZpJSB plasmid is arabinose-induced and glucose-repressedIn vivo characterization of Escherichia coli ftsZ MutantsCharacterization strategy = two different mediums + two different temperaturesFtsZFtsZJKD7-1 strain + pKD3 vector + pJSB vector containing a specific mutant FtsZTwo differe

9、nt CTwo different mediumsorInduction medium: LB + 0.05% arabinoseFtsZMutant FtsZFtsZRepression medium: LB + 0.2% glucoseXNo mutant FtsZFtsZ30 CWild type FtsZXFtsZ42 CNo FtsZFtsZFtsZ30 CRepression mediumPositive controlFtsZFtsZ42 CInduction mediumorcomplementNon-complementFtsZFtsZ30 CInduction medium

10、orNon-dominantnegativeDominant-negativeL269 doesnt complement and is dominant-negativeHydrophobic interactions are criticalHydrophilic interactions are not criticalAnswer to question #1:The observed inter-subunit interface is biologically relevantQuestion #2Whether the observed inter-subunit interfa

11、ce is a longitudinal interface or a lateral interface?GTPase assays revealed that both MtbFtsZ and EcFtsZ GTPase activities were dramatically affected by mutations at the interfaceMtbFtsZEcFtsZPolymerization studies revealed that mutations of the hydrophobic residues at the interface dramatically af

12、fect both MtbFtsZ and EcFtsZ protofilament formationsIn vivoIn vitroAnswer to question #2: the inter-subunit interface belongs to longitudinal interfaceWe observed a curved FtsZ protofilament conformationQuestion #3: What is the straight FtsZ protofilament conformation?MjFtsZ dimerOliva, et al 2004A

13、nswer to question #3:MjFtsZ dimer reflects a distorted longitudinal interfaceAnswer to question #3:SaFtsZ dimer represents a straight FtsZ protofilament modelSaFtsZ dimerTan, et al 2012A dramatic “straight to curved” conformational changeQuestion #4: How does this dramatic conformational change occu

14、r?T3 loop “T” to “R” stateconformational change T3 loop adopts two conformational states: tension (T) or relaxed (R)GTP-binding locks the T3 loop in T stateSaFtsZ dimerTan, et al 2012T3 loop in T state conformation is necessary for the assembly of straight protofilamentSaFtsZ dimerTan, et al 2012Mtb

15、FtsZ dimerThis studyConformational change from straight to curved can be interpreted as a hinge-opening event with the observed inter-subunit interface as a pivot point. The GDP bound between two subunits in SaFtsZ structure is completely occludedFour basic steps:1. Induction (Fuel enters)2. Compres

16、sion3. Ignition (Fuel is burnt)4. Emission (Exhaust out)Question #4:How is this conformational change converted to mechanical force?SummaryWe determined the first crystal structure of GDP-bound FtsZ protofilament in a curved conformational state.;The structure revealed an inter-subunit interface. Ou

17、r in vivo and in vitro studies showed this interface is biologically relevant and is part of the longitudinal interface;Our studies allow us to conclude that a recently determined Staphylococcal aureus FtsZ (SaFtsZ) structure represents a straight protofilament conformation;The curved MtbFtsZ protof

18、ilament therefore highlights a nucleotide-dependent conformational switch at the T3 loop that induces a “hinge-opening” event resulting in a dramatic longitudinal bending between FtsZ subunits after GTP hydrolysis.AcknowledgmentsLife Sciences Institute, Zhejiang UniversityYing LiYiwen ChengWeina ShangTsinghua Universi