Problems in Obtaining Diffraction-Quality Crystals of Integral Membrane Proteins Discussed in the context of 2 recently obtained structures of integral.

Problems in Obtaining Diffraction-Quality Crystals of Integral Membrane Proteins

• Discussed in the context of 2 recently obtained structures of integral membrane protein (IMP) complexes:

• (I) Hetero-oligomeric cytochrome b6f complex of oxygenic photosynthesis (8 gene products; dimer; 26 TM -helices; MW = 220 kDa); 3.0 Å.

• (II) Complex between the 22 strand -barrel E. coli outer membrane vitamin B12 receptor (BtuB) and the colicin E3 receptor (R) binding-domain; 2. 75 Å.

(II) The complex between the 22 strand -barrel vitamin B12 receptor and the colicin E3 R-domain.

(I) Cytochrome b6f complex: functions in membrane energy transduction

(I) The Cytochrome b6f Complexwith H. Zhang, G. Kurisu, & J. L. Smith

(II) Structure of the complex between BtuB and R135, which functions in protein import

40º

323313 438

447

LPSLPS

OM

(II) complex of the vitamin B12 receptor and the colicin E3 R-domain

with Genji Kurisu, Stas Zakharov, Masha Zhalnina, &M. Wiener, S. Bano, Y. Antonenko (not shown)

Challenge for Membrane Protein Structure Determination

Presently, in the protein data bank, there are > 22,000 protein structures. Among these,and 20 years after determination of thefirst integral membrane protein structure, there are 46 independent IMP structures, and 10 hetero-oligomeric IMP at a resolution 3.0 Å (http://www.mpibp.frankfurt. mpg.de/michel/public/memprotstruct.html).

Some problems in the crystallization of IMP1

• Use of thermophilic sources• Detergents: (i) undecyl-maltoside (); (ii) LDAO ()

• Purity; don’t over-purify! lipid depletion (part I).• Activity• Stability (oligomeric state; integral proteases)• Ligands for soluble domains (part II)• Problem of storage.

• 1 Iwata, S. (Ed.) [2003] Methods and Results in

Crystallization of Membrane Proteins., IUL, pp. 355

Electron transport complexes in oxygenic photosynthesis: cytochrome b6f complex provides electron connection between photosystem II & photosystem I reaction centers and translocates H+ across the membrane.

p (lumen) -side

n (stromal) -side

2H2O O2 + 4H+

PQ

4H+ PC (cyt c6)

Fd FNR Cyclic e-pathway

NADPH

PSII: Zouni et al

(2001) Nature 409,739 PSI: Jordan et al (2001) Nature 411, 909

Cyt b6f

Cells of the Thermophilic Cyanobacterium, Mastigocladus laminosus

Cross-Section of the Protein-Detergent Micelle Complex

Michel, H. (1990) Crystallization of Membrane Proteins;Pebay-Peyroula, et al., (1995) Structure, 3: 1051-1059

Electron Transfer Activity of Cytochrome b6f Complex

Efficiency of Action of Inhibitor

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

0 10 20 30 40 50 60 70

Vo

lta

ge

(V

)

Time (sec)

MLbf + DOPC

MLbf+DBMIB

MLbf

MLbf+DBMIB+DOPC

Masses of Eight Polypeptide Subunits of b6f Complex from the Thermophilic

Cyanobacterium, Mastigocladus laminosus

Whitelegge et al. Molec. Cell Proteomics (2002),1: 816-826

Subunit Measured Mass (Da)

Cyt f 32,270 Cyt b6 24,710 (calc., 24, 268)Rieske ISP 19,295Sub IV 17,529PetG 4057PetM 3841PetL 3530PetN 3304

Dimer MW = 217,057 Da

Two problems:

(i) It turned out that the protein was very pure, except for the possibility of trace protease (see below), and in fact was over-purified because the lipid was depleted (< 1 lipid/monomer);

(ii) the protease activity has not, until now,

been inhibitable.

Proteolysis Problem in First Crystals of the Cytochrome b6f Complex

1 2 3 4

Lane 1, fresh cytochrome b6f complex

Lane 2, after 7 days at room temperature Lane 3, crystalLane 4, thermolysin treated complex

Proteolysis of Cytochrome b6f Complex in Different Detergents

NG OG UDM DM DG DDM

t = 0

NG OG UDM DM DG DDM

t = 14 days

NG OG UDM DM DG DDM

t =7 days

Protease activity could not inhibited.

Crystals of cytochrome b6f complex from M. laminosus made after augmentation with the lipid,

DOPC (10:1, DOPC: Cytochrome f )

Hexagonal crystals, 78 % solvent content[Zhang, H. et al. (2003) PNAS, 100: 5160-5163]

SDS-PAGE of Cytochrome b6f Crystals

1 2 3Lane 1, fresh cyt b6f complex

Lane 2, new crystal

Lane 3, old crystal

Cyt f

Cyt b6

Sub IVISP

Cyt f

proteolysed Cyt b6

proteolysed ISP and Sub IV

Structure of Cytochrome b6f Complex

2 b-type Hemes, 1 c-type Heme, 1 [2Fe-2S]1 new heme, chlorophyll a, -carotene

p-side

n-side

DOPC

+10 kT-10 kT

Crystal Structure of the Complex between BtuB and R135 at 2.75 Å Resolution

40º

323313 438

447

LPSLPS

OM

Kurisu et al., Nat Struct Biol,

10: 948-954, 2003;pdb: 1UJW)

Problem of protein-protein contacts for “squat“ IMP in detergent; increase soluble domain with mab.

Hunte, C., H. Michel (2002) Curr Opin Struct Biol, 12: 503-508.

Cytotoxic colicins: colicin E3, a ribosomal RNAase;

n. b., coiled-coil motif

Colicin Ia Colicin N

Colicin E3

N

C

Domains:

Translocation

Receptor-binding

Activity

Mol Cell,8, 1053, 2001

Nature, 385, 461, 1997

To try to solve the problem of the lipid depletion, the purified complex was augmented with pure synthetic lipid.

• The result: the rate of formation of

crystals of intact complex increased greatly; i. e., crystals appeared over-night!

• Thus, the protease problem could be solved, but only by winning the race against it.

The E. coli Cell Envelope: receptor-containing outer-membrane, periplasmic space, & metabolically active inner-membrane

How are proteins imported across double membranes? Colicins as test molecules

How does colicin bind to, or insert into receptor?How does colicin bind to, or insert into receptor?n. bn. b., N-terminal cork (green) domain ., N-terminal cork (green) domain

blocks insertionblocks insertion

E. coliE. coli outer membrane protein BtuB, cobalamin outer membrane protein BtuB, cobalamin translocator, 22-antiparallel translocator, 22-antiparallel -barrel-barrel

((Chimento et al., Nat Struct Biol, 10, 394-401, 2003)

Colicin E3 receptor-binding domain (R135); Crystallization strategy: use R135 as soluble ligand of BtuB colicin receptor

Crystal Structure of the Complex between BtuB and R135 at 2.75 Å Resolution

40º

323313 438

447

LPSLPS

OM

Kurisu et al., Nat Struct Biol,

10: 948-954, 2003;pdb: 1UJW)

7-8

5-6

7-8

3-4

323

313 438

447

Two receptor translocon for colicin import across the E. coli outer membrane

Colicin E3

Cork domain

Acrobat Reader 5.0.lnk

AcknowledgmentsCytochome complex BtuB/R135 ComplexJ. T. Bolin Y. Eroukova (Moscow St.)A. Friedman M. LindebergD. W. Krogmann S. Schendel*M. Ponamarev R. Taylor*G. M. Soriano L. A. Sherman

DiscussionsM. G. Rossmann K. Jakes (AECOM)W. Minor (Virginia) M. Shoham (CWRU)

Synchrotron Lines & StaffAPS SBC-19 (N. Duke, F. Rotella); BioCARS 14 [Argonne NL]Spring-8 (Hyogo, Japan)

Grant SupportNIH-GMS (WAC); *NIH-GMS Biophysics Training Grant; Japan

Ministry of Science & Education (GK); DOE, NIH (APS)