BC530 Protein Structure II - UW Courses Web Servercourses.washington.edu/bioc530/2011_Lectures/2011...

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1 BC530 Protein Structure II “Seven Levels – part IIFall Quarter 2011 Wim G. J. Hol www.bmsc.washington.edu/WimHol PROTEIN STRUCTURE HIERARCHY THE SEVEN LEVELS 7 6 5 4 3 2 1 0 MULTI-MACROMOLECULAR ASSEMBLIES MULTI-SUBUNIT COMPLEXES MULTI-DOMAIN PROTEINS DOMAINS MOTIFS BASIC FOLDS CHAINS BUILDING BLOCKS 7 6 5 4 3 2 1 0

Transcript of BC530 Protein Structure II - UW Courses Web Servercourses.washington.edu/bioc530/2011_Lectures/2011...

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BC530

Protein Structure II“Seven Levels – part II”

Fall Quarter 2011

Wim G. J. Hol

www.bmsc.washington.edu/WimHol

PROTEIN STRUCTURE HIERARCHY

THE SEVEN LEVELS

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MULTI-MACROMOLECULAR ASSEMBLIES

MULTI-SUBUNIT COMPLEXES

MULTI-DOMAIN PROTEINS

DOMAINS

MOTIFS

BASIC FOLDS

CHAINS

BUILDING BLOCKS

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1. Morphological functionThe cell needs fibers, rings, cages

2. Cooperative functionBinding ligands in one subunit can affect conformation of other subunits in the complex – “allosteric proteins”

3. Stability against denaturation (???)This is probably a weak argument – small proteins can be very stable indeed – the authors have missed the point that not stability but functioning-with-flexibility is the key property of a protein in living organisms

4. Reduction of surface areaThe authors make an interesting case that smaller proteins have a larger surface and hence bind more water molecules per Dalton than multimeric proteins – since a cell contains about 20% protein in volume – proteins cannot all be small since then there would not be enough space left for water!

Goodsell and Olson give in their review the following reasons for building large protein assemblies:

D.S Goodsell and A J Olson “Functional Symmetry and Protein Function”Annu. Rev. Biophys. Biomol. Struct. 29, 105-153 (2000)

Proteins

Level 6:“MULTI-SUBUNIT PROTEINS”

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6.1 Multimers of identical subunits

6.1.1 Point group symmetry of which there are three types:- “cyclic”- “dihedral”- "cubic": tetrahedral, octahedral and icosahedral

6.1.2 Helical symmetryDefined by a rotation about the helix axis accompanied by a translation along that axis.

6.1.3 Non-symmetricSometimes the deviations from ideal symmetry are small and the term "pseudosymmetry" is used.

MULTI-SUBUNIT PROTEINS ("MULTIMERS")

Cyclic Point groups

Dihedral Point Group Symmetry

Cubic Point group Symmetry

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The Three Types of Point Group Symmetry

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C2 Dimers - Triosephosphate isomerase

- Lipoamide dehydrogenase

- and very many others

C3 Trimers - Influenza virus haemagglutinin

C4 Tetramers - Influenza virus neuraminidase

C5 Pentamers - The B subunits of cholera toxin

C6 Hexamers - Many, including ATPases

C7 Heptamers - GroES, a chaperonin

C11 Eleven-mers - Tryptophan RNA-binding Attenuation Protein

Multi-subunit proteins with Cyclic Point Group Symmetry

Be aware that not all Dimers of identical subunits need to have C2 Point group symmetry.

The two subunits can have different conformations; or are related by a non-twofold operation

A Dimer with Cyclic C2 Point Group Symmetry

Schematic

2 subunits

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A Heptamer with Cyclic C7 Point Group Symmetry

GroESThe “cap” of a protein folding machine

Side view Top view

Shekhar Mande

7 subunits

Multi-subunit proteins with Dihedral Point Group Symmetry

Definition: One "n-fold axis" with n 2-folds perpendicular to the n-fold.

All symmetry axes intersect in one point.

Examples:

D2(also called "222")

Tetramers - Hemoglobin (if considered identical to ß)- Glyceraldehyde phosphate dehydrogenase- many others

D3(also called "32")

Hexamers - Hemocyanin (arthropods)- Insulin

D4("42")

Octamers - Hemerythrin

D6("62")

Dodecamers - Glutamine synthase

D7("72")

14-mers - GroEL- Proteosome

D17 34-mers - Disks of Tobacco Mosaic Virus

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Schematic GroEL

A 14-mer with Dihedral D7 (or “72”)Point Group Symmetry

14 subunits

TETRAHEDRAL("T" or "23" symmetry)

12 subunits - Ferritin

OCTAHEDRAL("O" or "432" symmetry)

24 subunits - Cubic core of the PDC*- Small Heat Shock Protein

ICOSAHEDRAL("I" or "532" symmetry)

60 subunits - Dodecahedral Core of the PDC*- Riboflavin Synthase- Small Spherical Virus Capsids

Examples:

Multi-subunit proteins with Cubic Point Group Symmetry

(* PDC= Pyruvate Dehydrogenase Multi-enzyme Complex)

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Tetrahedral

Octahedral

Icosahedral

Concepts in Cubic Point Group Symmetry

Icosahedron

Dodecahedron

Octahedron

Hexahedron

Tetrahedron

The E2 core of the pyruvate dehydrogenase multi-enzyme complex (PDC)

60 subunits - viewed along one of the 30 twofold axesTina Izard

A 60-mer with Icosahedral (or “I”) Cubic Point Group Symmetry

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The E2 core of the pyruvate dehydrogenase multi-enzyme complex (PDC)

60 subunits - viewed along one of the twelve 5-fold axesTina Izard

A 60-mer with Icosahedral (or “I”) Cubic Point Group Symmetry

Examples

- Actin in e.g. muscleSteinmetz, M. O., Stoffler, D. & Hoenger, A. (1997).Actin: from cell biology to atomic detail.J. Struct. Biol. 119, 295-320.

- Tubulin in microtubulesNogales, E., Wolf, S. G., Khan, I. A., Ludueña, R. F. & Downing, K. H.Structure of tubulin at 6.5 Å and location of the taxol-binding site.

Nature 375, 424-426. (1995).

LEVEL 6.1.2

Multi-subunit proteins with Helical Symmetry

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Globular Actin, or G-Actin, is a four-domain protein of ~ 375 amino acid residues.It binds ATP which it can hydrolyze. It also binds calcium or magnesium.

Its most important property is to be able to assemble, and disassemble, into fibers,called microfilaments in non-muscle cells.

(Right-hand figure: The large gold sphere in the center indicates bound ATP: the small sphere the Mg ion)

Three-dimensional structure of an actin subunit

Steinmetz et al, J. STRUCTURAL BIOLOGY 119, 295–320 (1997)

Actin microfilament with helical symmetry

Steinmetz et al, J. STRUCTURAL BIOLOGY 119, 295–320 (1997)

Filtered electron microscopy image

Actin Subunitsinto e.m. image

Two intertwined strands

Two right-handed

intertwined strands

With 13 subunits per

turn, anda pitch of 71.5 nm,or 715 Å.

715 Å

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Notes:

A. IN MANY CASES ASSEMBLIES OF PROTEINS HAVE ONLY A TEMPORARY EXISTENCE.

For instance:

(i) Certain hemoglobins only form multimers when oxygenated and are monomers whendeoxygenated.

(ii) In signal transduction, DNA transcription, cell cycle regulation and many other key processesthe assembly and disassembly of multi-protein complexes is very carefully regulated.

B. THE COMPOSITION OF MULTIPROTEIN COMPLEXES CAN BE REGULATED IN A VARIETY OFWAYS.

For instance by:

• phosphorylation & dephosphorylation

• farnesylation and covalent attachment of other fatty acid containing groups

• binding of GTP and subsequent slow hydrolysis to GDP

LEVEL 6.2 Multi-subunit proteins with non-identical subunits

Examples:

6.2.1 Different subunits but still forming a symmetric particle

Heterotetramer 2ß2 (C2) - hemoglobinHeterotetramer 2ß2 (C2) - pyruvate dehydrogenase (E1) of the PDC

6.2.2 Different subunits with partial symmetry

AB5 heterohexamer - cholera toxin where the B pentamer has C5 symmetryHeterotrimer (2ß) - Lipoamide dehydrogenase (E3) dimer in complex with

one E2 binding domain (E2BD) of the PDC

6.2.3 Different subunits with no symmetry

Heterodimer (ß) - Ras-like protein (rap)•Ras binding domain Heterotrimer (2ß) - Human Growth Hormone•Receptor ComplexDimer of a 13-mer - Cytochrome c Oxidase 13 different subunitsRNA polymerase II - 10 different subunits without any symmetry

Multi-subunit proteins with non-identical subunits

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Hemoglobin: An α2β2-tetramer with C2 Symmetry

Glu 6 is the point of a key mutation changing HbA into HbS, leading to sickle cell anemia

B pentamer…

Titia SixmaView along 5-fold axis

Cholera Toxin: A Pentamer, plus…

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Titia SixmaThe A-subunit

…a Monomer, make…

Titia Sixma

Assembly of the AB5 holotoxin

A-subunitB-pentamer +

Cholera Toxin&

Enterotoxin

Functions:The B-subunit binds to human cell surface receptors.The A-subunit modifies a key human protein inside the cell.

Active Site

One of five Receptor

Binding Sites

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The Pyruvate Dehydrogenase Multi-enzyme Complex (PDC)

A dynamic multifunctional complex which is differently constructed in different organisms but always contains multiple copies of at least three different types of enzymes:

E1: the pyruvate dehydrogenasecontains the co-factor thiamine diphosphate (TDP)E1's can be 2 homodimers or 2ß2 heterotetramers

E2: the lipoylacetyl transferasethe catalytic domain of E2 forms the high symmetry core of the PDCThis can be a 24-mer or a 60-mer; differs per species.

E3: the lipoamide dehydrogenasecontains the cofactor FADE3's are always a dimer.

The simplest PDC's have a molecular weight of approximately 4 million Daltons.

The more complex PDC's, which incorporate additional proteins, have molecular weights of 9 to 10 million Daltons.

The PDC: A Complicated Combination of Symmetries

1. The E1 and E1ß subunits form E12ß2 heterotetramers, containing two TDP cofactors.

2. The E3 subunits form E3 2 homodimers, containing two FAD cofactors.

3. The essential lysine of the E2-lipoyl domain is lipoylated, i.e., is covalently modified to contain the lysine-lipoamide group.

4. The catalytic domains of E2 form trimers.

5. Eight of these E2 trimers form a hollow truncated cube.

6. Some E2 Binding Domains (E2BD) associate with E12ß2 heterotetramers to form E2BD• E12ß2 heteropentamers.

7. Some E2 Binding Domains (E2BD) associate with E3 homodimers to form E2BD•E3 heterotrimers.

By this time a creature has been obtained with:a highly-symmetric hollow core from which extend 24 octopus-like flexible arms, each of which has bound either an E1 heterotetramer or an E3 dimer, while at the end of these arms lipoyl domains frolic about and visit successively three active sites: those of E1, E2 and E3.

The assembly of a (yes) simple Pyruvate Dehydrogenase Complex (PDC)

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The E1 α2β2 heterodimer (center)

Arnthor Aevarsson

The first component of the PDC

A thiamine diphosphate (TDP)-dependent enzyme

The E3 dimer

The E3 dimer –lipoamide dehydrogenase – has C2 symmetry.

It contains one FAD cofactor per subunit (not shown). Bram Schierbeek

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E2: The multi-domain central subunit of the PDC

E2- domains:

=CD

PDC: Interactions of the E2-Binding Domain (E2BD) with E1 and E3

Quite counter intuitively, only ONE E2BD binds per E1-hetero tetramer, and also only ONE E2BD per E3-dimer

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E2: Trimers formed by the catalytic domain (CD)

The E2-CD trimer has C3 symmetry.

It contains no cofactor, but is a “CoA acetyl transferases” Andrea Mattevi

E2: Two trimers of the catalytic domain (CD)

These E2-CD trimers might be along the assembly pathway

to a higher level of organisation… Andrea Mattevi

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Andrea Mattevi

E2: Hollow Cube formed by 24 catalytic domains

View along fourfold.

There are another four E2-CD trimers in a layer behind this four trimers in the front

E2 Lipoyl Domain

12 E1- heterotetramers (blue) surrounding the Cubic Core made by 24 E2-catalytic domains (red)plus 12 E3-dimers (green) plus ultra-simplified E2-lipoyl domains (yellow)

PDC: Hollow E2CD Cube plus E1, E3 and E2-Lipoyldomains

E3 Dimer

E1 heterotetramer

Cubic core made up by 24 subunits of catalytic domain of

E2

Andrea Mattevi

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Aevarsson et al.. Nat. Str. Biol. 6, 785 (1999)

A very dynamic multi-enzyme complex in action..

PDC: in reality, nothing simple and, certainly, nothing static.

Multi-Macs are here defined as complexes of

proteins with other RNA and/or DNA.

Examples are:

• DNA-binding proteins: interacting intimately with its cognate DNA

• Telomerase: a protein-RNA complex which elongates telomeres

• Nucleosomes: Histone-DNA complexes in the chromosome

• Ribosomes: the protein synthesizing machinery

• Spliceosomes: RNA modifying complexes

• And really numerous other examples

LEVEL 7: MULTI-MACROMOLECULAR ASSEMBLIES

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Iron-dependent Regulator IdeR from M. tuberculosis

George Wisedchaisri

Four IdeR subunits forming two dimers surround the DNA duplexA transient complex – regulated by the concentration of iron in the cell

The Nucleosome

The nucleosome core particle consists of about 146 bp of DNA wrapped in 1.67 left-handed superhelical turns around the histone octamer, consisting of 2 copies each of the core histones H2A, H2B, H3 and H4.

Adjacent nucleosomes are joined by a stretch of free DNA termed "linker DNA" which varies from 10 - 80 bp in length depending on species and tissue type.

Liljas, Textbook of Structural Biology. (Fig. 3.21)

FIGURE 3.21 Left: A dimer of histone proteins H3 (blue) and H4 (light blue). Right: Nucleosomestructure. The octameric complex of histone proteins forms the center and the DNA is woundaround. The color scheme of the histone subunits in the core particle is the same as in Fig. 3.20(PDB: 1KX3).

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The 30 S particle of the bacterial ribosome schematic

E. coli ribosome

Small subunit (30S)

one rRNA:

of 1542 nt

(560 kDa)

plus

21 polypeptides

(together 370 kDa)

Liljas, Textbook of Structural Biology. (Fig. 8.15)

The small ribosomal subunit with the RNA domains illustrated from the 5’- to 3’- end by red, green, yellow and blue ribbons.

Most of the proteins are also marked.

The 50 S particle of the bacterial ribosome

A view of the large ribosomal subunit looking down the peptide exit tunnel (arrow).RNA in silver ribbon; proteins in color,.

E. coli ribosomeLarge subunit (50S)

Two rRNAs:one of 2904 nt

another of 120 nt(together 1.1 MDa)

plus

31 polypeptides (together 0.49 MDa)

Liljas, Textbook of Structural Biology. (Fig. 8.14)

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The 50 S particle of the bacterial ribosome

Proteopedia – 26 Oct 2011

A view of the small (left) and large (right) ribosomal subunits (With tRNA bound; upper center)

Proteopedia: “This is the Noller 2.8 Angstrom structure (2j00 and 2j01) with the A-site tRNA from the 5.5 Angstrom (1gix) fit back in with the portion seen at 2.8 Angstrom.”

Three major classes of proteins

1. Water-soluble proteins

2. Membrane proteins

3. Fibrous proteins

These seem very different, but actually the borders are not that sharp.

Membrane proteins can be thought of as water-soluble proteins with a greasy surface around their belt. Moreover, the vast majority of membrane proteins contain a large number of hydrophilic domains in addition to one, or more, transmembrane domains.

Some water-soluble proteins contain long -helical coiled coils (e.g., myosin) reminiscent of some types of fibrous proteins like α-keratin, or contain Pro-rich stretches with a collagen-like chain.

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The Structure of Fibrous Proteins

(From “From Cells to Atoms” - Rees and Sternberg)

Silk α-Keratin

Collagen

(Collagen Fig. from Finkelstein and Ptitsyn - “Protein Physics”; Data and Hyp from Voet,Voet,Pratt – Fundamentals of Biochemistry)

The Collagen Triple Helix

Collagen: the most common vertebrate protein

A single collagen molecule consists of three chains

Type I Collagen:

Two α1 and one α2 chains

Each ~ 950 amino acids long

Width: 14 Ǻ, length: ~ 3000 Ǻ.

About 30% of residues are Gly, 15-30% are 4-hydroxyPro

Repeat of Gly-X-Y , often Gly-Pro-Hyp.

Each individual chain is a LEFT handed helix.

Three of these helices are wound around each other with a gentle, RIGHT-handed twist.

A Gly at every third position in the chain makes it possible to bring the chains very close together in the center of the triple helix: The Gly NH makes an H-bond with the C=O of the Pro of an adjacent chain.

(Vitamin C is required for the enzyme making

4-hydroxyPro)

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Amyloids and Folding Diseases

Several diseases are now known to be caused by protein misfolding, in particular by forming insoluble fibrous aggregates known as amyloids.

Examples:

- Alzheimer’s disease

- amyloid plaques consisting mainly of amyloid β- protein

- Bovine spongiform encephalopathy (BSE or “mad cow disease”)

- aggregates of misfolded prion protein molecules

- Several amyloidoses, including familial amyloid polyneuropathy

- fibrous arrangements of misfolded transthyretin (a blood plasma protein, carrying hormones like thyroxine)

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A model of an amyloid fibril

Perhaps even more surprisingly is the fact that there are many examples of useful amyloids produced by organisms ranging from bacteria to humans.

(Otzen, Structure 19: 1207-1208 (2011)

Characteristics:

- the individual β-strands run perpendicular to the

direction of the fibril;

- adjacent β-strands are surprisingly tightly packed.

- this tight packing makes it difficult to

remove/digest these assemblies and hence they cause major and often fatal problems in and between cells.

Surprisingly, quite a few proteins appear to be able to adopt

such a type of arrangement of

β-strands arranged in life-threatening

fibers.

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Chapters and Books on Symmetry

Cantor & Schimmel - "Biophysical Chemistry"Part I: The Conformation of Biological MacromoleculesChapter 2-6: Quarternary Structure

"Symmetry - A Unifying Concept"by I. Hargittai & M. HargittaiAn easy-going introduction to symmetry

"The Geometrical Foundation of Natural Structures"by R. WilliamsParticularly good on polyhedra

"Multi-Protein Assemblies with Point Group Symmetry"by A. Kumar, A Ævarsson, W.G.J. HolIn “Perspectives in Structural Biology. A volume in honor of G.N. Ramachandran” (eds. Vijayan, M., Yathindra, N. & Kolaskar, A.S.) pp. 449-466 (1999)

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