SS 2008 lecture 9
Biological Sequence Analysis1
V9 Pharmacogenomics of P-Glycoprotein
Review of lecture V8 ..
SS 2008 lecture 9
Biological Sequence Analysis2
Inside the lipid bilayer, the protein backbone may not form hydrogen bonds with the aliphatic chains of the phospholipid molecules the backbone atoms need to form H-bonds among eachother.
Peptide segments crossing the membrane adopt either -helical or -sheet conformations.
Crash course on membrane protein structure:secondary Structure of TM proteins (V6 membrane bioinformatics)
SS 2008 lecture 9
Biological Sequence Analysis3
Only 2 architectures of Membrane Proteins
http://www.biologie.uni-konstanz.de/folding/Structure%20gallery%201.html
SS 2008 lecture 9
Biological Sequence Analysis4
History of membrane protein structure determination
1984 bacterial reaction center noble price to Michel, Deisenhöfer, Huber 1987
1990 EM map of bacteriorhodopsin Henderson
1997 high-resolution structure by Lücke
now several intermediates of the photocycle
1992 porin (complete -barrel)
1998 halorhodopsin
1995 Cytochrome c Oxidase
1998 F1ATPase noble price to John Walker 1997
1998 KCSA ion channel noble price to Roderick McKinnon 2003
2000 aquaporin
2000 rhodopsin (Palczewski)
2002 SERCA Ca2+ ATPase (Toyoshima)
2003 voltage-gated ion channel
2005 NaH Antiporter (Hunte)
SS 2008 lecture 9
Biological Sequence Analysis5
Lipid bilayer simplifies the prediction problem
TM proteins are forced into two classes: -helical, or -sheet.
-helices are typically tilted with respect to the membrane normal
between 10 – 45°.
The hydrophobic lipid bilayer reduces the three-dimensional structure formation
almost to a 2D problem.
SS 2008 lecture 9
Biological Sequence Analysis6
Predicting TM helix location
Hydrophobicity scales provide simple criteria to predict membrane helices.
TMH can be predicted based on the distinctive patterns of hydrophobic (TM) and polar (non-membrane or water-soluble) regions within the sequence.
Observed patterns:(1) TM helices are predominantly apolar and 12-35 residues long.
(2) Globular regions between TMH are typically shorter than 60 residues
(3) Most TMH proteins have a specific distribution of the positively charged amino acids arginine and lysine, „positive-inside-rule“ (Gunnar von Heijne). Connecting „loop“ regions on the inside of the membrane have more positive charges than „loop“ regions on the outside.
SS 2008 lecture 9
Biological Sequence Analysis7
Kyte-Doolittle hydrophobicity scale (1982)
Assign hydropathy value to each amino acid.
Use sliding-window to identify membrane regions. Sum the hydrophobicity scale over all w residues in the window of length w.
Use threshold T to assign segment as predicted membrane helix.
w = 19 residues could best discriminate between membrane and globular proteins.
Threshold T > 1.6 was suggested for the average over 19 residues.
SS 2008 lecture 9
Biological Sequence Analysis8
More refined indices
One drawback of pure hydropathy-based methods is that they fail to discriminate accurately between membrane regions and highly hydrophobic globular segments.
Other hydrophobicity scales:
- Wimley & White : based on partition experiments of peptides between water/lipid bilayer and water/octanol
- TMFinder (Liu & Deber scale) : based on HPLC retention time of peptides with non-polar phase helicity.
http://blanco.biomol.uci.edu/hydrophobicity_scales.html
SS 2008 lecture 9
Biological Sequence Analysis9
Folding of helical membrane proteins
White, FEBS Lett. 555, 116 (2003)
SS 2008 lecture 9
Biological Sequence Analysis10
Hydrophobicity Scales
White, FEBS Lett. 555, 116 (2003)
SS 2008 lecture 9
Biological Sequence Analysis11
Translocon-assisted folding of TM proteins?
White, FEBS Lett. 555, 116 (2003)
Upper picture (model!):the newly synthesized polypeptide chain of a membrane protein is inserted from the ribosome into the membrane via interaction with a TM complex, the “translocon” (EM map shown).
lower picture:experiment largely supports the concerted view.
What determines insertion into the membrane ?
SS 2008 lecture 9
Biological Sequence Analysis12
Integration of H-segments into the microsomal membrane
Hessa et al., Nature 433, 377 (2005)
b, Membrane integration of H-segments with the Leu/Ala composition 2L/17A, 3L/16A and 4L/15A. Bands of unglycosylated protein are indicated by a white dot; singly and doubly glycosylated proteins are indicated by one and two black dots, respectively.
Ingenious experiment! Introduce marker that shows whether helix segment H is inserted into membrane or not.
a, Wild-type Lep has two N-terminal TM segments (TM1 and TM2) and a large luminal domain (P2). H-segments were inserted between residues 226 and 253 in the P2-domain. Glycosylation acceptor sites (G1 and G2) were placed in positions 96–98 and 258–260, flanking the H-segment. For H-segments that integrate into the membrane, only the G1 site is glycosylated (left), whereas both the G1 and G2 sites are glycosylated for H-segments that do not integrate in the membrane (right).
SS 2008 lecture 9
Biological Sequence Analysis13
Insertion determined by simple physical chemistry
gg
g
fff
p21
1
g
gapp f
fK
2
1
Hessa et al., Nature 433, 377 (2005)
c, Gapp values for H-segments with 2–4 Leu residues.
Individual points for a given n show Gapp values obtained when the position of Leu is changed. d, Mean probability of insertion (p) for H-segments with n = 0–7 Leu residues.
measure fraction of singly glycosylated (f1g) vs. doubly glycosylated (f2g) Lep molecules
appapp KRTG ln
SS 2008 lecture 9
Biological Sequence Analysis14
Biological and biophysical Gaa scales
Hessa et al., Nature 433, 377 (2005)
a, Gappaa scale derived from H-segments with the indicated amino acid placed in
the middle of the 19-residue hydrophobic stretch.Only Ile, Leu, Phe, Val really favor membrane insertion. All polar and charged ones are very unfavored.
b, Correlation between Gappaa values measured in vivo and in vitro.
c, Correlation between the Gappaa and the Wimley–White water/octanol free
energy scale for partitioning of peptides.
SS 2008 lecture 9
Biological Sequence Analysis15
Positional dependencies in Gapp
Hessa et al., Nature 433, 377 (2005)
a, Symmetrical H-segment scans with pairs of Leu (red), Phe (green), Trp (pink) or Tyr (light blue) residues. The Leu scan is based on symmetrical 3L/16A H-segments with a Leu-Leu separation of one residue (sequence shown at the top; the two red Leu residues are moved symmetrically outwards) up to a separation of 17 residues. For the Phe scan, the composition of the central 19-residues of the H-segments is 2F/1L/16A, for the Trp scan it is 2W/2L/15A, and for the Tyr scan it is 2Y/3L/14A. The G app value for the 4L/15A H-segment GGPGAAALAALAAAAALAALAAAGPGG is also shown (dark blue). b, Red lines show G app values for symmetrical scans of 2L/17A (triangles), 3L/16A (circles), and 4L/15A (squares) H-segments. c, Same as b but for a symmetrical scan with pairs of Ser residues in H-segments with the composition 2S/4L/13A.
Tyr and Trp are favorable in interface region.
SS 2008 lecture 9
Biological Sequence Analysis16
MINS
Park & Helms, Bioinformatics 24, 1271 (2008)
Amino acid frequencies across the membrane found in X-ray structures of TM proteins (19 positions centered at the membrane center) were interpreted as indicator of their insertion free energy.
To convert frequencies into free energies, they were calibrated against exp. values for Hessa peptides.
Plot of MINS-predicted and experimentally measured membrane insertion free energies for 357 known cases
SS 2008 lecture 9
Biological Sequence Analysis17
Using grammatical rules
The lipid bilayer constrains the structure of the membrane-passing regions of proteins in many ways.
TMHMM (Sonnhammer et al. 1998, Krogh et al. 2001) and HMMTOP (Tusnady & Simon 1998, 2001) implement Hidden Markov Models.
TMHMM: uses cyclic model with 7 states for - TM helix core- TM helix caps on the N- and C-terminal side- non-membrane region on the cytoplasmic side- 2 non-membrane regions on the non-cytoplasmic side (for short and long loops to account for different membrane insertion mechanism)- a globular domain state in the middle of each non-membrane region
SS 2008 lecture 9
Biological Sequence Analysis18
Summary of TM secondary structure
TM helices are typically continuous stretches of mostly hydrophobic residues.
Simple methods based on summing up hydrophobicities work okay but not really well.
Advanced methods include additional features such as the „positive-inside rule“.
The currently most successful methods are based on Hidden Markov Models or Neural Networks.
Evaluating performance accuracy should be done using carefully separated training and test sets.
It is possible to discriminate signal peptides and TM helices.
Only Split 4.0 may detect short non-membrane spanning helices.
SS 2008 lecture 9
Biological Sequence Analysis19
Positioning of TM proteins in membrane(from V7 membrane bioinformatics)
In the absence of high-resolution 3D structures, an important cornerstone for the functional analysis of any membrane protein is an accurate topology model.
Topology model: describes the number of TM spans and the orientation of the protein relative to the lipid bilayer.
Topology models can be generated by sequence-based prediction or by experimental approaches.
SS 2008 lecture 9
Biological Sequence Analysis20
Idea: generate reference point, e.g. the location of a protein‘s C terminus.
In E.coli attach alkaline phosphatase (PhoA) that is active only in the periplasm of E.coli, or green fluorescent protein (GFP) that fluoresces only in the cytoplasm.
(1) Global Topology Analysis
Daley et al. Science 308, 1321 (2005)
SS 2008 lecture 9
Biological Sequence Analysis21
Functional categorization of E.coli inner membrane proteome
Daley et al. Science 308, 1321 (2005)
clear trend for Nin – Cin topologies (even number of TMH)- largest functional category is transport proteins, many with 6 or 12 TM helices. Most proteins with unknown function have 6 TM helices.
SS 2008 lecture 9
Biological Sequence Analysis22
Most TM proteins are expected to adopt only one topology in the membrane.Global topology analysis of E.coli inner membrane proteome identified 5 dual-topology candidates: EmrE, SugE, CrcB, YdgC, YnfY.All are quite small (~ 100 aa), contain 4 strongly predicted TM segments, contain only few K and R residues and have very small (K + R) bias.
(2) Dual-topology proteins?
Rapp et al., Nat.Struct.Biol. 13, 112 (2006)
(a) A dual-topology protein inserts into the membrane in two opposite directions. As nearly all helix-bundle membrane proteins have a higher number of lysine (K) and arginine (R) residues in cytoplasmic (in) than in periplasmic (out) loops (the ‚positive-inside‘ rule), dual-topology proteins are expected to have very small (K + R) biases.
Rectangles: TM segmentsblack dots: K and R residues
SS 2008 lecture 9
Biological Sequence Analysis23
Without solving their 3D structures, how can one prove that a protein has dual topology?
Such a protein would be particularly sensitive to the addition or removal of a single positively charged residue in a loop or tail. measure activities of two different, C-terminally fused reporter proteins:PhoA (only enzymatically active when in the periplasm)GFP (fluorescent only when in the cytoplasm).
Concentrate on N-terminus and first loop.
Dual-topology proteins?
Rapp et al., Nat.Struct.Biol. 13, 112 (2006)
SS 2008 lecture 9
Biological Sequence Analysis24
(a) wt YdgE-PhoA fusion is active,wt YdgE-GFP fusion is inactive C-terminus in periplasm (Cout )
wt YdgF behaves oppositely (Cin)
These 2 proteins are topologically stable.
(b – d) C-terminal orientation of EmrE, SugE, CrcB, YnfA and YdgC is highly sensitve to charge mutations. For 14 or 19 charge mutations, both PhoA and GFP activities change in the direction expected from the change in (K + R) bias.
Charge mutations shift the orientations of dual-topology TM proteins
Rapp et al., Nat.Struct.Biol. 13, 112 (2006)
SS 2008 lecture 9
Biological Sequence Analysis25
Experimental techniques to study orientation of proteins in membraneschemical modificationspin-labelingfluorescence quenchingX-ray scatteringneutron diffractionelectron cryomicroscopyNMRpolarized infrared spectroscopy.
Desirable to complement by computational methods.
e.g. explicit-solvent molecular dynamics... up to simplified approaches that minimize the protein transfer energyfrom water to a hydrophobic slab (used as a membrane model).
(3) Positioning of proteins in membranes
Adamian & Liang, Proteins 63, 1 (2006)
SS 2008 lecture 9
Biological Sequence Analysis26
important parameters
Lomize et al. Prot.Sci. 15, 1318 (2006)
SS 2008 lecture 9
Biological Sequence Analysis27
Model protein as a rigid body that freely floats in the planar hydrocarbon core of a lipid bilayer.
Calculation of transfer energy
Adamian & Liang, Proteins 63, 1 (2006)
ii
MW
iitransfer zfASAdzG ,,, 0
ASAi : accessible surface area of atom i, computed with NACCESS
iW-M : solvation parameter of atom i (transfer energy of the atom from water to
membrane interior in kcal/(mol.Å2) )
f(zi): interfacial water concentration profile with = 0.9 Å
0
11
zzi i
ezf
SS 2008 lecture 9
Biological Sequence Analysis28
ionization of charged residues
Residues that are typically charged in soluble proteins may become neutral in the hydrophobic inside of the bilayer!
The ionization/protonation energies of charged residues are described by the Henderson-Hasselbalch equation:
Lomize et al. Prot.Sci. 15, 1318 (2006)
aioniz pKpHRTG 3.2
at pH = 7
average pKa value Gioniz
in proteins [kcal/mol]Arg 12.0 6.9Lys 10.4 4.7Asp 3.4 4.9Glu 4.1 4.0His 6.6 0.6
SS 2008 lecture 9
Biological Sequence Analysis29
use deterministic search strategy:(1) grid scan to determine a set of low-energy combinations of variables z0, d, ,
(2) local energy minimization (Davidon-Fletcher-Powell method) starting from low-energy points
Also consider energetically best rotation of solvent-exposed charged side chains (e.g. Lys and Arg) that are situated close to the calculated boundaries and could be rotated away from the hydrophobic core
Global energy optimization
Adamian & Liang, Proteins 63, 1 (2006)
SS 2008 lecture 9
Biological Sequence Analysis30
Average tilt angles
(a) hydrophobic thickness is also determined by optimization procedure.Computed values match experimental values well
Lomize et al. Prot.Sci. 15, 1318 (2006)
(b) the calculated helix tilt angles are in excellent agreement with NMR data,they also correlate well with ATR-FTIR data (table 3)
SS 2008 lecture 9
Biological Sequence Analysis31
Determine membrane segments
Lomize et al. Prot.Sci. 15, 1318 (2006)
SS 2008 lecture 9
Biological Sequence Analysis32
Peripheral and monotopicproteins have low penetrationdepths.
Calculated tilt angles vary from 0° - 6°. TM proteins tend to be nearly perpendicular to the membrane, although the individual helices are on average tilted by 21°.
Application to membrane proteins
Lomize et al. Prot.Sci. 15, 1318 (2006)
SS 2008 lecture 9
Biological Sequence Analysis33
Biological membranes differ
Lomize et al. Prot.Sci. 15, 1318 (2006)
SS 2008 lecture 9
Biological Sequence Analysis34
Ion channels and pumps(from V9 membrane bioinformatics)
Life’s chemistry of aqueous solutions employs ions as carriers of cell signals.
Such a signal is the action potential. That such a simple all-or-nothing signal should require highly complex proteins and ion channels, rather than just a particle within membrane bilayer, was unknown when Hodgkin and Huxley first described the processes of activation and inactivation of cation currents during the action potential in the early 1950s.
More than conveying rapid excitation by action potentials alone, other internal cell processes are initiated by ion signals.
Accordingly, the expression of these proteins is not restricted to excitable cells such as neurons or muscle but can be observed in external and internal membranes of almost all cells.
Lehmann-Horn, Jurkat-Rott, Physiol. Rev. 79: 1317-1372, 1999
SS 2008 lecture 9
Biological Sequence Analysis35
Channel function and structure
Ion-conducting membrane channels are opened by ligands or voltage changes (usually depolarization) and closed by a delayed inactivation that is simultaneouslyinitiated with the activation.
Sustained exposure to the ligand or the depolarization may lead to reopenings of the channel if the circumstances (time, voltage) allow the channel to recovery from the inactivated state.
The ion conducting pore is highly selective for a specific ion as in most voltage-gated channels, or it conducts cations or anions without high selectivity as in most ligand-gated channels.
The structures of the pore, its selectivity filter, and its activation and inactivation gates show high evolutionary conservation that allows one to make deductionson structure-function relationships from one channel type to the next.
Lehmann-Horn, Jurkat-Rott, Physiol. Rev. 79: 1317-1372, 1999
SS 2008 lecture 9
Biological Sequence Analysis36
potassium channels
Fig. shows X-ray structures of all available KC channels overall channel architecture is well conserved.The extracellular side is at the top, and the intracellular side is at the bottom. Main structural elements: outer helix, pore helix, selectivity filter and inner helix.There are 3 K+ ions, 2 of them are located at S1and S3 position in selectivity filter and 1 is at the center of the cavity.
SS 2008 lecture 9
Biological Sequence Analysis37
Pore in the middle between four proteins.The determination of the structure of the KcsA KC channel by X-ray crystallography provided the first atomic-resolution view of these proteins.
KcsA channel: tetrameric organization
Doyle et al. Science 280, 69-77 (1998)
Roderick MacKinnon,Nobel price in chemistry, 2003
SS 2008 lecture 9
Biological Sequence Analysis38
KCSA channel: selective conduction of potassium ions highly conserved pore region in sequence
Pore region highly conserved
Multiple sequence alignment of various potassium channels.
Doyle et al. Science 280, 69-77 (1998)
SS 2008 lecture 9
Biological Sequence Analysis39
Structure of KcsA potassium channel
B. Roux, Ann. Rev. Biophys. Biomol. Struct. 34, 153 (2005)
Most important functional feature of channel structure: 12Å long narrow pore located along the tetrameric symmetry axis near the extracellular side. Lined exclusively by main-chain carbonyl oxygen atoms from the residues corresponding to the signature sequence TTVGYG common to all KC channel, this region of the protein acts as a “selectivity filter” by allowing only the passage of nearly dehydrated K+ ions. Short alpha-helices from each of the four subunits, referred to as the pore helices, surround the selectivity filter with their COOH termini pointing toward the center of a wide aqueous cavity, about 15Å in diameter and able to contain 25 to 30 water molecules. It has been suggested that this aqueous cavity, located at the center of the membrane, helps overcome the electrostatic barrier to ion translocation that is opposed by the low dielectric membrane lipid.
SS 2008 lecture 9
Biological Sequence Analysis40
function
Doyle et al. Science 280, 69-77 (1998)
Selectivity filter
Helix-dipoles stabilizeion in central cavity.
Opening of channelentrance can be switched.
SS 2008 lecture 9
Biological Sequence Analysis41
B. Roux, Ann. Rev. Biophys. Biomol. Struct. 34, 153 (2005)
Single-file transport mechanism in KcsA
SS 2008 lecture 9
Biological Sequence Analysis42
TM-Protein with complicated structure.
During ion transport, protein undergoes conformational transitions between 2 states E1 and E2.
SERCA calcium pump
Toyoshima et al. Nature 405, 647-55 (2000)
SS 2008 lecture 9
Biological Sequence Analysis43
Superimposition of protein in 2 X-ray conformations.
SERCA Calcium-Pumpe
Pumping of single ion requires gigantic conformational changes.
Toyoshima et al. Nature 418, 605-11 (2002)
SS 2008 lecture 9
Biological Sequence Analysis44
C. Toyoshima, Nature 432, 361 (2004)
Conformational changes during catalytic cycle
A cartoon depicting the structural changes of the Ca2 -ATPase during the reaction cycle, based on the crystal structures in five different states.
SS 2008 lecture 9
Biological Sequence Analysis45
C. Hunte et al., Nature 435, 1197 (2005)
Na/H antiporter
Overall architecture of NhaA.12 TMSs are labelled with roman numerals. N and C indicate the N and C termini
Na+/H+ antiporters have primary functions in the regulation of intracellular pH, cellular N+ content and cell volume. They are integral membrane proteins that are ubiquitous throughout all biological kingdoms. NhaA is the main Na+/H+ antiporter of E. coli and many other enterobacteria. Its orthologues are widespread in many other prokaryotes. NhaA uses the electrochemical proton gradient maintained across the bacterial membrane and excretes N+ in exchange for a ‘downhill’ flow of protons into the cell. NhaA activity is strictly regulated by pH, a property it shares with many other prokaryotic and eukaryotic antiporters and which is essential for cytoplasmic pH regulation. At acidic pH NhaA is downregulated.
SS 2008 lecture 9
Biological Sequence Analysis46
C. Hunte et al., Nature 435, 1197 (2005)
Proposed mechanism of pH regulation and translocation
The TMSs IV/XI assembly, the charge-compensating residues D133 and K300, and further structural elements involved are shown schematically. a, Acidic pH-locked conformation. Ion transport is prevented by the periplasmic ion barrier (transparent, cream-coloured area) and by the only partly exposed Na+ (Li+ )-binding site (residues D164, D163, T132). b, Activation by alkaline pH induces conformational changes in helix IX resulting in the reorientation of helices IVc and XIp (interactions indicated by blue dotted lines). The putative Na+ (Li+)-binding site (yellow transparent circle) is now exposed to the cytoplasmic funnel (red dotted lines and red circle) and sealed towards the periplasm (orange bar).c, Na+ (Li+) binding results in the opening of the periplasmic funnel and the exposure of the active site to the periplasm. The cation is released. Protonation of the aspartates (D164 and D163) brings the antiporter back to the active conformation open to the cytoplasm.
SS 2008 lecture 9
Biological Sequence Analysis47
ABC-transporters
ABC-transporters utilize the energy of ATP hydrolysis to transport various substrates across cellular membranes. In bacteria, ABC-transporters mainly pump essential compounds such as sugars, vitamins, and metal ions into the cell.In eukaryotes, ABC-transporters mainly transport molecules to the outside of the plasma membrane or into membrane-bound organelles (ER, mitochondria, etc.).
www.wikipedia.org
Lipid flippase MsbA
Vitamine B12 transporter-like ABC transporters, BtuCD
Molybdate transporter
AB2C2 complex, open state
The range of transported compounds includes:Lipids and sterols Ions and small molecules. Drugs Large polypeptides.
SS 2008 lecture 9
Biological Sequence Analysis48
P-Glycoprotein
P-glycoprotein (abbreviated as P-gp or Pgp) is a well-characterized human Abc-Transporter of the MDR/TAP subfamily.
It is extensively distributed and expressed in normal cells such as those lining the intestine, liver cells, renal proximal tubular cells, and capillary endothelial cells comprising the blood-brain barrier.
P-gp is also called ABCB1, ATP-binding cassette sub-family B member 1, MDR1, and PGY1.
Sakurai et al., Biochemistry, 46, 7678 -7693, 2007