BMB 170 Lecture 15Lipids and Membranes, Nov 14, 2017

• Lipids• Bilayers• Membrane proteins

• Membrane protein structure websites:– Stephen White lab at UC Irvine

• “Membrane Proteins of Known 3D Structure)• http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html

S.J. Singer’s fluid mosaic modelSinger & Nicolson Science (1972) 175, 720-31 Lipid/Protein ratios

myelin 3disk memb(eye) 1E. coli 0.4purple memb 0.2

Integral membrane proteins and the phospholipid bilayer

Fig 4.27

Membrane Proteins - Overview• Lots of processes

– transport and transduction processes that mediate the flow of matter– energy and information across the membrane bilayer

• Poorly characterized relative to water-soluble proteins– experimental challenges of mimicking the membrane and water-

bilayer interfaces

• Constitute an estimated ~20-30% of all proteins, yet only <1000 distinct structures are available (116 10/06; 133 10/07; 174 10/08; 204 10/09; 261 12/10; 722 10/2017)

• ~50% of drug targets

• Corollary - lots of opportunities for research

doubling time ~ 3 years

Tuesday, Nov 15, 2016: 115374 (105570 last year) protein structures

Membrane protein types

Apolar membrane interior

2 kinds of secondary structure• α-helical - found in

cytoplasmic membranes and ER derived organelles

• β-sheets - all bacterial outer membrane proteins and outer membranes of mitochondria and chloroplasts

• The need is to satisfy the hydrogen bonding capabilities of peptide bonds

Bacterial export

Review: Wickner & Scheckman (2005) Science 310:1452

Protein translocation channel (1rhz)

• Universally conserved• Passive conduit• Opens in two directions• Hydrophilic interior• Recognizes signal sequence• Sets topology

Review: Clemons et al Curr Op Struct Bio (2004)14(4):390-6van den Berg et al Nature (2004) 427:36-44

Model for translocation

(+)

(-)

Modeling Translocation

P. Tian and I. Andricioaei (2006) Biophys J

16Å diameter ball can be pulled through the

channel.

See also J. Gumbart and K. Schulten (2006) Biophys J

Co-translation complex

Becker..Beckmann (2009) Science 326:1369

Mammalian Ribosome/Sec61 complex to 3.4Å

Voorhees, Fernández, Scheres, Hegde Cell (2014)157:1632-43

Other membrane insertasesYidC Structure

Kumazaki..Nureki Nature (2014) 509:516 (3wo6)

TatC Structures

Ramasamy..Clemons Structure (2013) 21:777 (4hts) Rollauer..Lea, Berks Nature (2012) 492:210 (4b4a)

Membrane partitioning governed by hydrophobicity• Ideal TM helices were tested

for membrane insertion with single amino acid substitutions

• Strong preferences for certain amino acids

• Membrane insertion purely driven by hydrophobicity

Review: White & von Heijne Curr Op Str Bio (2005) 15:378-86von Heijne Lab: Hessa et al Nature (2005) 433:377-81

N=window size (~21)

Hydropathy analysis of TM helices

White Lab, UC Irvinehttp://blanco.biomol.uci.edu/hydrophobicity_scales.html

http://www.cbs.dtu.dk/services/TMHMM-2.0/

von Heijne lab: Krogh et al. JMB (2001) 305:567-80

Active role for translocon?

• Partitioning model only works at steady state

• Translocon must open in relation to hydrophobic peptide

Zhang & Miller (2010) PNAS 107:5399

Removing proteins• Essential process• Bacterial components

somewhat understood• Much more complicated

in eukaryotes– Linked to many diseases– Deshaies lab

Akiyama (2009) J Biochem 146:449

Positive inside rule

• Noted by von Heijne

• TMs generally have a more positive charge on the cytoplasmic side

von Heijne (1986) EMBO J 5:3021

Genome TM prediction

von Heijne lab: Krogh et al. JMB (2001) 305:567

P. falciparum (Malaria) 2 chromosomes

• Histograms of predicted numbers of TM helices from different genomes

• Orientation preferences– Nin is favored– Nin/Cin favored

Global topology of E. coli inner membrane

• Used GFP and PhoA C-terminal fusions to establish topology

• 737 genes possible identified 601

• Data strengthens TMHMM models

• A few examples of dual topology!

• Using homology can identify 30% of all bacterial proteins (51K/660K)

• Extended it to Yeastvon Heijne lab: Daley et al Science (2005) 308:1321-3

Kim et al PNAS (2006)103(30):11142-7

Cin

Cout

Inverted repeat domains

• Common feature of some membrane proteins

• Evolutionary implications– Small Multidrug Resistant

transporter family (4TM)– Bacterial/Archael

Transporter family(5TM)– Drug/Metabolite Exporter

family (10TM)

Pornillos & Chang FEBS Letters (2006)580:358-62

YdgE/YdgF

EmrE

Route of integration

van Lehn..Miller (2015) eLifealso Woodall, Yin & Bowie (2015) Nature Comm

Glycine zippers (GXXXGXXXG) are strongly overrepresented in TM helices and provide a strong driving force for right-handed helical packing

Kim et al. PNAS (2005)102:14278; Lemmon et al. Biochemistry(1992) 31:12719

“Glycine” zippers at helix-helix interfaces

Affects of proline kinks

WT P50A

• In rhodopsin studies by Bowie et al– P50A mutation did not

remove kink– ΔΔGu = +0.1 kcal/mol

Evolution link to kinks• Not all kinks have

prolines• Proteins have lost

prolines but kept kinks

XHel

ix

P

P

Compensating Mutations

Modern day Proline kink

Y

Modern day non-proline kink

Proline peaks

80 aligned rhodopsins

40 aligned reaction centers

10 MPs (Bowie lab):

• 39 total kinks

• 22 kinks at proline

• 17 non-proline kinks. Of these, 14 show a peak of prolines in the alignment

• No proline peaks not associated with a kink

PRC Chain M: Proline frequency in 40 aligned sequences

0

5

10

15

20

25

30

35

40

45

50

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

Residue Number

Num

ber

of P

rolin

es

A147 Kink 13o

A153 Kink 34o

P165 Kink 49o

T277 Wide turn 35o

Rhodopsin: Proline frequency in TM Helices from 80 aligned sequences

05

1015202530354045505560657075808590

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310

Residue Number

Num

ber

of P

rolin

es

P303 Kink 46o

P53 Kink 20o

T92 Wide

turn 48o

P215 Wide turn 34o

P267 Kink 44o

P291 Kink 20o

A117 Kink 20o

GPCR classes show different patterns

0102030405060708090

30 50 70 90 110 130 150 170 190 210 230 250 270 290 310

Num

ber

of P

rolin

es

Rhodopsin

Residue Number

A B C D E F G

0

10

20

30

40

50

60

70

120 145 170 195 220 245 270 295 320 345 370 395

Num

ber

of P

rolin

es

Secretin

A B C D E F G

Interfacial preference for Tyr and Trp (esp. in β-barrel MPs)

20 Å

Cowen et al Nature (1992)358:727-33 (1pho)Review: Schultz Curr Op Struct Bio (2000) 10:443-7

Polar aromatics at the interface

“…the hydrophobic force is the energetically dominant force for containment, adhesion, etc., in all life processes. This means that the entire nature of life as we know it is a slave to the hydrogen-bonded structure of liquid water.”

proteinwater

Charles Tanford Protein Science (1997) 6:1358-66

What happens when a protein leaves water (membrane proteins)?

-1 0 +1

Asp Ile

hydrophobicity

buriedsurfacewater-soluble proteins

buried / surfaceTM region, 16 α-helical mps

Spencer & Rees Annu Rev Biophys Biomol Struct (2002) 31:207-33Rees et al Science (1989) 245:510-3

Hydrophobicity scale: Eisenberg et al Faraday Symp Chem Soc (1982) 17:109-20

Hydrophobicities of buried/surface residues

In table, relative packing efficiency is the occupied volume of atoms relative to well packed soluble structures. Lower than 100% indicates a tighter packing.

Gerstein & Chothia Science (1999) 285:1682-3

Packing efficiency of buried atoms

Diacylglycerolkinase in detergent unfolding by CD

Two state model for analysis of SDS denaturation

∆G = 16 kcal/mole for membrane part

Temp dependence still unknown

• much more difficult to study (irreversible) (PROBABLY NOT TRUE ANYMORE…)

• denatured state is not completely unfolded (helices remain intact)

• detergents often destabilizing

• stability-enhancing mutants– common, unlike soluble proteins– Bowie COSB (2001)11:397-402

Membrane Protein Stability

Lau & Bowie Biochemistry (1997)36:5884

Faham et al JMB (2004)335:297-305

Bacteriorhodopsin B helixAlanine scanning to test contribution to stability

Surface residues make relatively small contribution to protein stability

destabilizingsomewhat destabilizingminimal affectstabilizing (25%)

Membrane protein stability

water membrane

• membrane proteins and water soluble proteins have similar– interior apolarities– packing densities– surface areas– patterns of residue conservation– stabilities

• and differ in – surface polarities– helix-helix packing distributions– tertiary folds

Membrane protein - water-soluble protein comparison

Slovic et al. PNAS (2004) 101:1828

Water-solubilization of KcsA

• Designed computationally then made it – confirmed binding to known inhibitors

• Different surface hydrophobicity for different solvents - same internal packing

• Water soluble proteins are membrane proteins with built-in detergent

Reviewed in Borgese et al (2007) COCB 19:368

Tail-anchored protein targeting

Cyanobacterial Get3Nostoc. PCC 7120 All4481 (3IGF)

S. cerevisiae Get3 (2WOJ)

Synechocystis PCC 6803,Hohmann-Marriott et al. 2009

Fusion

Organelle fusion

Review: Wickner & Schekman (2008) NSMB 15:658

• low pH of endosomes induces conformational change

• Eventual coiled-coil leads to fusion

Wiley & Skehel labs: Bullough et al Nature (1994) 371:37-43

Fusion peptide Membrane

fusion

Dengue low pH fusion

Harrison lab: Modis et al Nature (2004) 427:313-9 (1ok8)

This fusion is a general feature of viruses Class I: myxo/paramyxo (flu/measles), retroviruses (HIV), filoviruses (Ebola)Class II: flaviviruses (Dengue & West Nile) and alphaviruses (Semliki Forest)

low pH

C

Fusion Peptide

Lipid Anchoring

• Cytosolic face– Fatty acid

• Myristoyl (C14)– Amino group of N-terminal

glycine• Palmitoyl (C16)

– Cys residue – regulated?– Prenylation

• Polyisoprenoid– Farnesyl (C15) or geranylgeranyl

(C20)– Modified C-terminal Cys (CXXY)

• Outside– Glycosylphosphatidylinositol

• GPI- anchor• Most common in • Amide linkage to C-terminal

residue of protein• Lipid raft localization

GPI Review :Paulick & Bertozzi (2008) Biochemistry 47:6991