Bioinformatics & Bioinformatics & Structural BiologyStructural Biology

M93360008生技所 研一 劉怡萱

Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol

☆ JBC 2004 Oct.

Jason Gunawan1, Dave Simard2, Michel Gilbert3, Andrew L. Lovering1, Warren W. Wakarchuk3,Martin E. Tanner2, Natalie C. Strynadka1

Mass Spectrometry

☆ ☆ Analysis of Tertiary and Quaternary Analysis of Tertiary and Quaternary Structure of Sialic Acid Synthase by ChStructure of Sialic Acid Synthase by Chemical and MS methodemical and MS method

OH

O

HO

OH

OHNH

O

HO

O

OH

Sialic acid

Sialic acids, a family of 3-deoxy-2-keto nine Sialic acids, a family of 3-deoxy-2-keto nine carbon sugars are located in the non-reducing tercarbon sugars are located in the non-reducing terminal ends of cell-surface glycoconjugates of virminal ends of cell-surface glycoconjugates of virus, mammalian cells, and some bacteria.us, mammalian cells, and some bacteria.

The geneThe gene neuB neuB encodes the enzyme, encodes the enzyme, sialic acid synthase, catalyzing the condesialic acid synthase, catalyzing the condensation of ManAc with PEP to give sialic nsation of ManAc with PEP to give sialic acid.acid.

Streptococcus aglactiaeStreptococcus aglactiae“Characterization of Escherichia coli Sialic Acid Synthase” Biochem. Biophys. Rapid Comm.2002, 295, 16

☆ Sialic acid & sialic acid synthase

sialic acid synthase

Product

☆ Evidence of Dimeric Dimer

SDS-PAGE

DMA

8.6 Å

DMS

11.0 Å

DMA : Dimethyl adipimidate

DMS : Dimethyl suberimidate·2HCl

728.0 1377.8 2027.6 2677.4 3327.2 3977.0

Mass (m/z)

0

4.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity

Voyager Spec #1=>BC=>NR(2.00)[BP = 885.8, 49272]

885.7871

1715.38301290.1202

1642.2404

2325.9595

1559.2245

1574.31521083.0467 1610.2678 2454.1025

1835.36621270.1181861.3429

2946.39931596.31152171.67391353.11671105.0574877.3227 1857.3638

1454.12281233.1622 2458.0667 3466.7270841.3423 2199.6286 2968.05501820.5681 3196.1843

728.0 1377.8 2027.6 2677.4 3327.2 3977.0

Mass (m/z)

0

4.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity

Voyager Spec #1=>BC=>NR(2.00)[BP = 885.8, 49272]

885.7871

1715.38301290.1202

1642.2404

2325.9595

1559.2245

1574.31521083.0467 1610.2678 2454.1025

1835.36621270.1181861.3429

2946.39931596.31152171.67391353.11671105.0574877.3227 1857.3638

1454.12281233.1622 2458.0667 3466.7270841.3423 2199.6286 2968.05501820.5681 3196.1843

728.0 1377.8 2027.6 2677.4 3327.2 3977.0

Mass (m/z)

0

4.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity

Voyager Spec #1=>BC=>NR(2.00)[BP = 885.8, 49272]

885.7871

1715.38301290.1202

1642.2404

2325.9595

1559.2245

1574.31521083.0467 1610.2678 2454.1025

1835.36621270.1181861.3429

2946.39931596.31152171.67391353.11671105.0574877.3227 1857.3638

1454.12281233.1622 2458.0667 3466.7270841.3423 2199.6286 2968.05501820.5681 3196.1843MVYI I AE I GCNHNGDINLAKKMVDVAVSCGVDAVKFQTFK AEKLISKFAPKAEYQKATTGTADSQLEMTKRLELSFEEYLEMRDYAISKGVETFSTPFDEESLEFLISTDMPIYKIPSGE ITNLPYLEKLGKQQKKVILSTGMAVMEEIHQAVNILRQNG TTDISILHCTTEYPTPYPSLNLNVIHTLKDEFKDLTIGYSDHSIGSEVPIAAAAMGAEVIEKHFTLDTNMEGPDHKASAT PDILAALVKGVRIVEQALGRFEKIPDPVEEKNKIVARKSV VALKPIKKGDIYSIENITVKRPGNGISPMNWYDILGQEAQ DDFEEDEVIRDSRFENQLPELHHHHHH

Introduction to Peptide Mass Fingerprinting

In solution In solution digestiondigestion

In gel digestionIn gel digestion

MALDI-TOF MS MALDI-TOF MS AnalyzeAnalyze

Database searchDatabase search

peptide fragmentsintact protein

proteolysis

GDIYSIENITVK

DEESLEFLISTDMPIYK

ITNLPYLEK

In solution tryptic digestion

5min

10min

20min

M/Z

M/Z

M/Z

☆ The time resolved limited proteolysis and differential MALDI-TOF

MS mapping

MALDI- MS

MALDI- MS

MALDI- MS

347

89 116 157 223 288 334N

5min

10min

20min

30min

Over night

☆ The time resolved limited proteolysis and differential MALDI-TOF MS mapping

SUBTRACT

Tryptic digestion

Chemical cleavage

☆Identification of Cross-Linked Peptides in the Interface Regions

Unmodified peptides

Surface-labeled peptides

Intramolecular cross-linked peptides

Putative intermolecular cross-linked peptides

m/z

Inte

nsity

Inte

nsity

m/z

N

O

O

OO

OO

O

O

O

O

N

O

O

M.W 456.36

Spacer Arm 16.1Ǻ

O

O

O

O

O

O

O

O

O

O

O

O

O

OOH

O

O

O

O

++

COMPLEX EGS ( a NHS ester)

INTERMOLECULAR

+ 226 Da

INTRAMOLECULAR

+ 226 Da

HYDROLYSED

+ 244 Da

-NH

-NH

-NH2 -NH2

-NH

-NH

-NH

+228

-1 -1

+245

-1

Crosslinking

☆ Chemical cross-linking by EGS

N

O

O

- N

O

O

-

N

O

O

N

O

O

-++

NO

O

+82Da+164Da

+82Da+82Da

650 1320 1990 2660 3330 4000

Mass (m/z)

0

3.8E+4

0

10

20

30

40

50

60

70

80

90

100

% Intens

ity

Voyager Spec #1 MC=>BC[BP = 1811.8, 37587]

1811.8470

A

7

52735

2327

B

1339

15

163338

3538

32 3926

C D

34

H

I

20 25J

NML

K

1

2

3

4

6 8 10

9

1112

14

18

19

22

24

30

31

36

37 402332

Rel

ativ

e in

tens

ity(%

)

Cleaved fragments of intermolecular Cleaved fragments of intermolecular cross-linked peptidecross-linked peptide

Cleaved fragments of intramolecular croCleaved fragments of intramolecular cross-linked peptidess-linked peptide

☆ Mapping interface peptides by MALDI-TOF

650 1320 1990 2660 3330 4000

Mass (m/z)

0

2.5E+4

0

10

20

30

40

50

60

70

80

90

100

% Intens

ity

Voyager Spec #1 MC=>BC[BP = 1811.8, 25061]

1812.8527

1

2

3 46

9

11

12

14

19

22

30

36

3740

X5

X

7

810

X13

X15

16X X

17

18 2021

23 24

25

X26

27

28

X29 31

32

33X

34 35 38 39

M/Z

Rel

ativ

e in

tens

ity(%

)

XX

Intermolecular cross-linked peptideIntermolecular cross-linked peptide

Intramolecular cross-linked peptideIntramolecular cross-linked peptide

Hydrolysed peptideHydrolysed peptide

Unmodification peptideUnmodification peptide

☆ Proposed Interface Regions of NeuB

# Observed mass Residue [M+H]+ assignments

27 2520.3 278-287-EGS-278-28832 3013.6 44-51-EGS-36-51

33 3069.3 41-51-EGS-57-7135 3180.8 278-287-EGS-116-13238 3707.4 57-71-EGS-116-13239 3810 261-273-EGS-261-277 FEK IPDPVEEorK NK + FEK IPDPVEEorK NorK IVAR

KSVVALK PIK + IPSGEITNLPYLEK LGKATTGTADSQLEMTK R + IPSGEITNLPYLEK LGK

LISK FAPK + FQTFK AEorK LISorK FAPK

AEK LISorK FAPK + ATTGTADSQLEMTK R

KSVVALK PIK + K SVVALKPIKK

Sequence assignments

44-51 57-71

36-51 57-71 116-132

261-273 278-287

261-277 278-287

N

N

347C

347C

NeuB domain Antifreeze-like Domain256

Proposed interface

Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol

☆ JBC 2004 Oct.

Jason Gunawan1, Dave Simard2, Michel Gilbert3, Andrew L. Lovering1, Warren W. Wakarchuk3,Martin E. Tanner2, Natalie C. Strynadka1

☆ The eight-stranded a /b barrel (TIM barrel)

☆ The importance of ligands

☆ ☆ Multiple Sequence Alignments of NeuB familyMultiple Sequence Alignments of NeuB family

☆ ☆ Multiple Sequence Alignments of NeuB from Multiple Sequence Alignments of NeuB from Neisseria Meningitidis and Streptococcus aglactiae

SaNeuB 1 ------------------MVYIIAEIGCNHNGDINLAKKMVDVAVSCGVDAVKFQTFKAEKLISKFAPKAN.NeuB 1 MQNNNEFKIGNRSVGYNHEPLIICEIGINHEGSLKTAFEMVDAAYNAGAEVVKHQTHIVEDEMSDEAKQV

SaNeuB 53 EYQKATTGTADSQLEMTKRLELSFEEYLEMRDYAISKGVETFSTPFDEESLEFLISTDMPIYKIPSGEITN.NeuB 71 IPGNADV----SIYEIMERCALNEEDEIKLKEYVESKGMIFISTPFSRAAALRLQRMDIPAYKIGSGECN

SaNeuB 123 NLPYLEKLGKQQKKVILSTGMAVMEEIHQAVNILRQNGTTDISILHCTTEYPTPYPSLNLNVIHTLKDEFN.NeuB 137 NYPLIKLVASFGKPIILSTGMNSIESIKKSVEIIREAG-VPYALLHCTNIYPTPYEDVRLGGMNDLSEAF

SaNeuB 193 KDLTIGYSDHSIGSEVPIAAAAMGAEVIEKHFTLDTNMEGPDHKASATPDILAALVKGVRIVEQALGRFEN.NeuB 206 PDAIIGLSDHTLDNYACLGAVALGGSILERHFTDRMDRPGPDIVCSMNPDTFKELKQGAHALKLARGGKK

SaNeuB 263 KIPDPVEEKNKIVARKSVVALKPIKKGDIYSIENITVKRPGNGISPMNWYDILG-QEAQDDFEEDEVIRDN.NeuB 276 DTIIAGEKPTKDFAFASVVADKDIKKGELLSGDNLWVKRPGNGDFSVNEYETLFGKVAACNIRKGAQIKK

SaNeuB 332 SRFENQLPELN.NeuB 346 TDIE------

☆ ☆ the sturcture of S.NeuB sould be similar to N.NeuBthe sturcture of S.NeuB sould be similar to N.NeuB

☆ Homology modelong of Sa NeuB

347

89 116 157 223 288 334N

5min

10min

20min

30min

Over night

☆ The time resolved limited proteolysis and differential MALDI-TOF MS mapping

347

89 116 157 223 288 334N

347

89 116 157 223 288 334N

347

89 116 157 223 288 334N

A subunit 44-51

B subunit 57-71

B subunit 36-51

44-51 57-71

36-51 57-71 116-132

261-273 278-287

261-277 278-287

N

N

347C

347C

A subunit 116-132

B subunit 57-71

44-51 57-71

36-51 57-71 116-132

261-273 278-287

261-277 278-287

N

N

347C

347C

B subunit 116-132

A subunit 278-287

44-51 57-71

36-51 57-71 116-132

261-273 278-287

261-277 278-287

N

N

347C

347C

A subunit 261-277

B subunit 261-277A subunit 278-287

B subunit 278-287

44-51 57-71

36-51 57-71 116-132

261-273 278-287

261-277 278-287

N

N

347C

347C

H7

H8

H7

H8

KK89

115

KK

157RR

222KK

288KK

333RR

KK

K

K K

K

KK

K

K

K

K

K

KK

K

K

K

K

K

K

K

KK

H7

H8

☆ the sturcture of S.NeuB sould be similar to N.NeuB

☆ the imperfection of the method of “ time resolved limited proteolysis time resolved limited proteolysis ”

☆ does the tetramer real exist ?

☆Conclusion