XIII International Congress of the Spanish Biophysical Society

Antimalarial Activity of Cupredoxins:The Interaction of Plasmodium MSP119 and Rusticyanin

Irene Díaz-Moreno

Structural Bases of Mitochondrial Dysfunctionin Response to Cytochrome c Phosphorylation

at Tyrosine 48

Irene Díaz-Moreno

Thr28Tyr48

Tyr97

Ser47

Lee et al. (2006) Biochemistry 45, 9121-8

Hüttemann et al. (2012) Adv. Exp. Med. Biol. 748, 237-64Hüttemann et al. (2012) Biochim. Biophys. Acta – Bioenergetics 1817, 598-609

Hüttemann et al. (2011) Mitochondrion 11, 369-81Yu et al. (2008) Biochim. Biophys. Acta – Bioenergetics 1777, 1066-71

Cc is Phosphorylated in vivo

Zhao et al. (2011) Molec. Cell. Proteomics 10, M110.000299

Guerra-Castellano et al. (2016) Biochim. Biophys. Acta - Bioenergetics 1857, 387-95

Pecina et al. (2010) Biochemistry 49, 6705-14García-Heredia et al. (2011) J. Biol. Inorg. Chem. 16, 1155-68

Tyr48

Respiratory Function

Cardiolipin Peroxidation

Apoptosis

Lee et al. (2006) Biochemistry 45, 9121-8

Phosphorylated alters Cc function

Traditional tyrosine-by-glutamate substitution

GlutamatePhosphotyrosine

≠

García-Heredia et al. (2010) Biochim. Biophys. Acta – Bioenergetics 1797: 981-93

García-Heredia et al. (2012) FEBS Lett. 586: 154-8

Díaz-Moreno et al. (2013) ChemPhysChem 14: 3095-102Díaz-Moreno et al. (2012) Chem. Eur. J. 18: 3872-8

Díaz-Moreno et al. (2011) Biochim. Biophys. Acta – Bioenergetics 1807: 1616-23García-Heredia et al. (2011) J. Biol. Inorg. Chem. 16: 1155-68

Tyrosine-by-pCMF substitution

p-carboxymethyl-L-phenylalanine(pCMF)Phosphotyrosine

Dittmann et al. Germany. Patent number: ES 2366469 T3. 2011-03-11Mischra & Hamburger. (1993) Cancer Res. 53: 557–63

Advantages of pCMF:

• Stable

• Resistant to protein tyrosinephosphatase hydrolysis

Potential used of pTyr:

• Cytoprotective on non-tumoral cells

• Inhibitor of tumoral cells

≈

Tyrosine-by-pCMF substitution

p-carboxymethyl-L-phenylalanine(pCMF)Phosphotyrosine

≈

Guerra-Castellano et al. (2015) Chem. Eur. J. 21: 15004-12

Evolved tRNA Synthetase Technique

pCMF

+Unnatural

Aminoacyl- tRNAsynthetase

ATPP P P

AUC

pCMF tRNA

P P

AUC

UAG5’ 3’AGC

Nascentpeptide

mRNAAUCUCG

pCMF chargedtRNA

Guerra-Castellano et al. (2015) Chem. Eur. J. 21: 15004-12

10 9 8 7

130

125

120

115

110

105

100

15N

(ppm

)

1H (ppm)6

57

31

49

51

79 47

48

89

61

2

88 5981

2783

80

104

82

90

18

100

6973

835

10374

91

33

1517

70

102

2924

68 19

6

77

4113

37 36

345423

98

9516

11

64

99

3 2610

92101

5262

637514

78

4060

20

72

50

8622 43

387

9632 55

469

651242

2597 4

55867

53

9439

56

87

85

66

93

Y48pCMF Cc

WT Cc

PDB file: 2N3Y; BMRB dataset: 25660

Y48pCMF Cc NMR Structure

Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

PDB file: 2N3Y; BMRB dataset: 25660

by CYANA / RMD

Y48pCMF Cc NMR Structure

Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

pCMFmutation

loop

Y48pCMF Cc NMR Structure

PDB file: 2N3Y; BMRB dataset: 25660

by CYANA / RMD

Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

Heavy atomsSide-chain Atoms

Y48pCMF Cc NMR Structure

PDB file: 2N3Y; BMRB dataset: 25660Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

WT Cc

Y48pCMF Cc

Difference

PDB file: 2N3Y; BMRB dataset: 25660

NMR Dynamics by HetNOE

Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

Unaffected

Undetectable NH signals

NOE and R1

R2

Heme grouppCMF

PDB file: 2N3Y; BMRB dataset: 25660

Y48pCMF Cc NMR Dynamics

Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

Cytochrome c: Functions in celular metabolism

Martínez-Fábregas et al. Mol. Cell. Proteomics. 12 (2013) 3666–3676Martínez-Fábregas et al. Mol. Cell. Proteomics. 13 (2014) 1439–1456

Martínez-Fábregas et al. Cell Death Dis. 5 (2014) e1314González-Arzola et al. PNAS 112 (2015) 9908–9913

Cytochrome c as electron carrier

Louro & Díaz-Moreno, eds. (2015) Redox Proteins in Supercomplexes and SignalosomesMoreno-Beltrán et al. (2014) Biochim. Biophys. Acta. 1837: 1717–29

Moreno-Beltrán et al. (2015) FEBS Lett. 589: 476–483

Rcf1/2 Rcf1/2

Residues withCSPs (Δδavg):

› 0.075

0.050 - 0.075

0.025 - 0.050

< 0.025 Unassigned and ProlinesHeme group

Y48pCMF Cc

WT Cc

Y48pCMF Cc Interaction with Cc1

180o

Moreno-Beltrán et al. (2015) FEBS Lett. 589: 476–483Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

Respiratory Supercomplexes

NADH

NAD

FADH2 FADCI

CII CIIICIV

Q pool

Cc

½O2 + 2H+ H2O

IMM

FADH2 FADNADH

NAD

IMM

½O2 + 2H+ H2O

Dependent on metabolic needs

Acín-Pérez, R. et al. Mol. Cell 32 (2008) 529–539

Y48pCMF cytochrome c as an electron carrier

YPD: Supports fermentation and respirationYP-Gal: Supports respiration. Supplementedwith 0.5% of lactic acid.

WT CcY48pCMF Cc

Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

Model of ETC interactions

Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

Cytochrome c: Functions in celular metabolism

Martínez-Fábregas et al. Mol. Cell. Proteomics. 12 (2013) 3666–3676Martínez-Fábregas et al. Mol. Cell. Proteomics. 13 (2014) 1439–1456

Martínez-Fábregas et al. Cell Death Dis. 5 (2014) e1314González-Arzola et al. PNAS 112 (2015) 9908–9913

Activation of caspase-3 by Y48pCMF cytochrome c

Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

Y48pCMF cytochrome c

Moreno-Beltrán, Guerra-Castellano et al. (2017) PNAS Plus in press

1. The non-canonical amino acid pCMF emulates better that glutamatethe volume of phosphotyrosine.

pCMF is a stable analogue of a phosphotyrosine and is resistant to hydrolysis by tyrosine phosphatases.

2. Tyr48 phosphorylation of cytochrome c induces changes on proteindynamics at different timescales and the formation of secondaryconformations.

Take Home Messages

3. Phosphorylated cytochrome c shows a lower respiration rate thannon-modified protein in mitochondrial supercomplexes.

Mitochondrial OxPhos is regulated by phosphorylation of cytochrome c, allowing rapid adjustment

of the hemeprotein function to cellular changes.

4. Phosphorylated cytochrome c behaves as an efficient peroxidaseenzyme and an anti-apoptotic switch.

Biomolecular Interaction Platform (BIP)

http://bip.ciccartuja.es/home/

EU-COST Action (2016-2021)MOBIEU (CA 15126)

SBE International Congress — Seville 6-8 June 2017

http://sevilla2017.sbecongress.org/

Irene Díaz-MorenoBiointeractomics Unit

THANK YOU!Collaborators• Rita Bernhardt – Saarbrucken• Isabella Felli & Paola Turano – Florence• Myriam Gorospe – Baltimore, USA• Pau Gorostiza - Barcelona• Juan Hermoso – Madrid • Peter Hildebrandt – Berlin • B. Göran Karlsson – Göteborg• Abelardo López-Rivas – Seville• M. Luz Martínez-Chantar & Francisco Blanco – Bilbao• Pedro M. Nieto – Seville• Enrique Pérez-Payá & Mar Orzáez - Valencia• Andres Ramos – London• Marcellus Ubbink – Leiden• José M. Valpuesta – Madrid • Adrián Velázquez – Saragossa• Jackie Wilce – Clayton, Australia

XIII International Congress of the Spanish Biophysical Society

Antimalarial Activity of Cupredoxins:The Interaction of Plasmodium MSP119 and Rusticyanin

Irene Díaz-Moreno

Structural Bases of Mitochondrial Dysfunctionin Response to Cytochrome c Phosphorylation

at Tyrosine 48

Irene Díaz-Moreno