Topic Summaries

PDB Codes for structures discussed in lecture: PDB Protein 1PRC Photosynthetic reaction center 1GOX Glycolate oxidase 1MOB Myoglobin 1LDM Lactic dehydrogenase 1PYK Pyruvate kinase 8RUC Rubisco I have 1AUS for Rubisco 5NUL Flavodoxin 1AKY Adenylate kinase 1ROP Rop protein 2MHR Myohemerythrin 1HGU Human growth hormone 3HHR Human growth hormone + receptor 2SOD Superoxide dismutase 1BRP Retinol binding protein 1NSB Neuraminidase 2BNA DNA 1AON GroEL/GroES 1QLX Prion 1LHO Leucine Zipper 1ZAA Zinc finger 3GAP Catabolite activating protein -CAP 2RH1 GPCR-beta adrenergic receptor 3SN6 beta adrenergic receptor-Gs complex 1IGT Immunoglobulin 7GCH Chymotrypsin 2BRD Bacteriorhodopsin 2OMF Porin

I don’t recall learning about:

• 1LDM

• 5NUL

• 1AKY

• 1ROP

• 2MHR

1GOX: Glycolate Oxidase

1MOB: Myoglobin

1LDM: Lactic Dehydrogenase

1PYK: Pyruvate Kinase

5NUL: Flavodoxin

1AKY: Adenylate Kinase

1ROP: Rop Protein

3MHR: Myehemerythrin

1MBO: Oxymyoglobin -helix only

1GOX: Enzyme Glycolate Oxidase 8-barrel

1AUS: RUBISCO / motif

2SOD: Zn Super Oxide Dismutase Only s ; anti-paralle -barrel

2SOD: Zn Super Oxide Dismutase Only s ; anti-paralle -barrel

1BRP or 1BRQ: Retinol Binding Protein Antiparallel -barrel of 8 strands

1NSB: Neuroaminidase 1subunit showing organization of six 4-stranded sheets, together forming a propellar

1AON: GroEL/GroES complex

1AON: GroEL/GroES complex Bottom view

1HLO: Leucine Zipper

3GAP: CAP – Catabolite Activating Protein

1ZAA: Zinc Finger

1QLX: Prion

2BNA: DNA

2BRD: Bacteriorhodpsin

2OMF: Porin

2OMF: Porin, side view

4H33: K+ Channel

4H33: K+ Channel, side view

2OMF: Porin, bottom view

1PRC: Photosynthetic Rxn Cntr

2RH1: human beta2-adrenergic GPCR

1IGT: Immunoglobulin

1AO7: TCR + MHC

1AO7: TCR (blue/pink) MHC (white/yellow)

7GCH: Chymotrypsin

3SN6: B-Adrenergic R 1HGU: Human Growth Hormone

3HHR: Human GH + Rec

Papers = dialogue with scientists: Questions: Why? How? What? So what? Structure: to explain function Knowledge: for farming, food, fine chemicals, molecular medicine diagnosis / therapy Bread and Butter of Course: Structure determines function (You can intervene or control at this junction) How? Does structure determine function? Process: description of structure Dissection of function

The uniqueness of water as a biological solvent: Influences all molecular interactions. Why? b/c of: • Polarity (charge) • Cohesiveness (order) • Hydro/dehydration reactions • Proton transfer reactions • Origin of cavities = water free microenvironments Importance of Hydrogen Bonds: • Source of unique properties of water • Structure and function of proteins • Structure and function of DNA • Structure and function of polysaccharides • Binding of substrates to enzymes • Binding of hormones to receptors • Matching of mRNA to tRNA Levels of Structure in Protein Architecture Sequence Structure Function Primary: amino acid sequence Secondary: Folding motifs (α,β) Tertiary: Packing of secondary structures domains Quaternary: Packing of several polypeptide chains

Sequence / Structure Relationship:

Motif suited to create hydrophobic cavities

• Learn to use PDB • Protein molecules talk to each by SURFACES. • The protein has its chemistry on its SURFACE. • The chemistries are exposed by the protein generated by the

protein’s 3-D structure. • The structure of the protein depends on the a.a. sequence • Language of proteins is the language of biochemistry. • KNOW: names of a.a., their properties • w/o proteins, no function in cell!! a.a. Peptide, and Proteins Learning goals: • Structure and naming of a.a. • Structure and properties of peptides • Ionization behavior of a.a. and peptides • Methods to characterize peptides & proteins

Proteins = main agents of biological function • Catalysis

– Enolase (in the glycolytic pathway) – DNA polymerase (in DNA replication)

• Transport – Hemoglobin (transports O2 in blood) – Lactose permease (transports lactose across the cell mbn)

• Structure – Collagen (connective tissue) – Keratin (hair, nails, feathers, horns)

• Motion – Myosin (muscle tissue) – Actin (muscle tissue, cell motility)

a.a. = building blocks of proteins

• Proteins are linear heteropolymers of -amino acids

• Amino acids have properties that are well-suited to carry out a variety of biological functions

– Capacity to polymerize

– …acid-base properties

– …physical properties

– …chemical functionality

Most alpha-a.a. are chiral

• The -carbon always has four substituents and is tetrahedral

• All (except proline) have

– An acidic carboxyl group

– A basic amino group

– An -hydrogen connected to the -carbon

• The fourth substituent (R) is unique

– In glycine, the fourth substituent is also hydrogen

Amino Acide – atom naming

• Organic nomenclature: start from one end

• Biochemical designation:

– Start from -carbon and go down the R-group

Amino Acid Classification

• Common a.a. can be placed in five basic groups depending on their R substituents

– Nonpolar, alipathic (7) GAPVLIM

– Aromatic (3) FYW

– Polar, charged (5) STCNQ

– Positively charged (3) KRH

– Negatively charged (2) DE

Glycine vs. Proline Glycine has no side chain and therefore can adopt phi and psi angles in all four quadrants of the Ramachandran plot. Hence it frequently occurs in turn regions of proteins where any other residue would be sterically hindered.

• Proline shows only a very limited number of possible combinations of phi and psi.

Secondary Structures

• Secondary structure refers to a local spatial arrangement of the

polypeptide backbone

• Two regular arrangements are common:

– The -helix

• Stabilized by the H-bonds between nearby residues

– The -sheet

• Stabilized by H-bonds between adjacent segments that

may not be nearby

• Irregular arrangement of the polypeptide chain is called the random coil

(Loops)

-Helix • Helical backbone is held together by H-bonds between the C=O of a.a. (n)

and the N-H group of the a.a. four residues up the helix (n+4) • Right handed helix with 3.6 residues (5.4A) per turn • Peptide bonds are aligned roughly parallel with the helical axis • Side chains point out and are roughly perpendicular with the helical axis. - Helix dipole • Recall that the peptide bond has a strong dipole moment

– Carbonyl O negative – Amide H positive

• All peptide bonds in the -helix have a similar orientation • The -helix has a large macroscopic dipole moment • Negatively charged residues often occur near the positive end of the helix

dipole. Sequence affects Helix Stability • Not all polypeptide sequences adopt -helical structures • Small hydrophobic residues such as Ala and Leu are strong helix formers • Pro acts as a helix breaker b/c the rotation around the N-Ca bond is

impossible • Gly acts as a helix breaker b/c the tiny R-group supports other

conformations. -Sheets • The planarity of the peptide bond and tetrahedral geometry of the -

carbon create a pleated sheet-like structure • Sheet-like arrangement of backbone is held together by H-bonds

between the backbone amides in different strands • Side chains protrude from the sheet alternating in up and down direction. Parallel and anti-parallel -Sheets • Parallel or anti-parallel orientation of two chains within a sheet are

possible. • In parallel sheets the H-bonded strands run in the same direction

– Resulting in bent H-bonds (weaker) • In anti-parallel sheets the H-bonded strands run in opposite directions

– Resulting in linear H-bonds (stronger) Turns

• turns occur frequently whenever strands in sheets change the direction

• The 180 turn is accomplished over a spread of four a.a.

• The turn is stabilized by a H-bond from a carbonyl oxygen to amide proton three residues down the sequence

• A Proline in position 2 or glycine in position 3 are common in turns.

Reversible interactions in Mol Bio

Eletrostatic ~3Å 3-7 kcal/mol

Hydrogen Bonds ~3Å 3-7 kcal/mol

Van der Waals <3Å ~1 kcal/mol

Covalent Bond (C-C) 1.5Å ~80 kcal/mol

Photon (green light) ~60 kcal/mol

Helix Dipole

Motifs Simple combination of secondary structure elements with specific geometric array • Examples of Motifs

– Helix-loop-helix – β-loop-β – β-Hairpin (common in parallel β-sheets) – β-α-β (common in parallel β-sheets) – Helical Bundles -- are combinations of α-helices and short loops; # of

bundles vary, the packing of “knobs into holes” is an important feature for achieving stability

Specific arrangement of several secondary structure elements

– All -helix

– All -sheet

– Both

• Motifs can be found as reoccuring structures in numerous proteins

• Proteins are made of different motifs folded together

Topological Diagrams

• Domain – a unit, a polypeptide that independently folds into tertiary structure

• α/β - most frequent of regular protein structure

• Different sequence functions

/ Motifs

Closed = Barrel made of 8+8 (8alpha helices + 8 beta strands); active site formed by loops at the C-end of the beta sheet

Open = twisted sheet made of variable alpha helices and beta strands; active site formed by functional residues provided by the loop regions that connect the C-end of -strand to N-end of -helix

• Parallel or mixed -sheet with helices on both sides

• Number of -strands vary from 4 to 10

• Called also Rossman fold

-Barrels: Antiparallel β- Strands

Functions of beta barrels:

– Binding-Retinol binding protein

– Transport-Porin

– Catalysis-SOD

– Recognition-Antibodies

Structures: (motifs)

– Up and down (cavity)

– Greek key (regular)

– Jelly roll (distorted)

• Fold: β-helix – wide helix with 2 or β-strands/turn

Orazio Notes:

Types of Beta Barrels

• Up-and-down beta barrel: Up-and-down barrels are the simplest barrel topology and consist of a series of beta strands, each of which is hydrogen-bonded to the strands immediately before and after it in the primary sequence. (Ex. PORINS, RETINOL)

• Greek key: Greek key barrels have some beta strands adjacent in space that are not adjacent in sequence. Beta barrels generally consist of at least one Greek key structural motif linked to a beta hairpin, or two successive Greek keys. N-type and also C-type (ex. DOS)

• Jelly roll: The jelly roll barrel, also known as the Swiss roll, is a complex nonlocal structure in which four pairs of antiparallel beta sheets, only one of which is adjacent in sequence, are "wrapped" in three dimensions to form a barrel shape. (ex.

• Structures we studies in class:

– Retinol Binding Protein; DOS; PORINS Influence Virus (RNA) • Membrane Enveloped • NA – Neuraminidase (Tetramer); HA – Hemaglutinin (Trimer) Example of Molecular Parntership • HA Binds to sialic acid on surface of suceptible cells endocytosis RNA release • NA cleaves sialic acid, assists in releasing progeny virions Neuroaminidase (NA)

• 1600 a.a.

• Four subunits

• 24 - strands/subunits (superbarrel)

• 6 motifs: 4 -strands per motif (up and down)

• Active Site: formed by loops at one end of the super barrel

– Wide, funnel-shaped

Super Oxide Dismutase 8-stranded "Greek key" beta-barrel, with the active site held between the barrel and two surface loops. The two subunits are tightly joined back-to-back, primarily by hydrophobic and some electrostatic interactions.

The ligands of the copper and zinc are six Histidine and one aspartate side-chains; one Histidine is shared between the two metals.

Porins…are beta barrel proteins that cross a cellular membrane and act as a pore through which molecules can diffuse

α/β Motifs # of β-Strands Active Site

α/β Closed Barrel 8+8 Loops at C-end of β-sheet

α/β Open

Twisted Sheet =

Parallel β-Strands

Variable

Crevice at C- end of β- Strand opposite of β- Sheet

Active site is formed by functional residues provided by the

loop regions that connect C-end of β- with the N-end of α

Rossman Fold = twisted beta sheet

Super Oxide Dismutase Porin

DNA

• Most DNA molecules are double-stranded helices, consisting of two long polymers of simple units called nucleotides, molecules with backbones made of alternating sugars (deoxyribose) and phosphate groups , with the nucleobases (G, A, T, C) attached to the sugars.

• The two strands run in opposite directions to each other and are therefore anti-parallel, one backbone being 3' (three prime) and the other 5' (five prime).

• Components: Bases (AGCT); Sugar (deoxyribose); Phosphate group. All together form a nucleotide

• Directionality: 5 prime to 3 prime. Location of H bonds.

• History: Watson and Crick. Why they thought about the helical shape, what did they use? Chargaff rule. Franklin: X ray diffraction image of DNA.

• DNA periodicity: 34 and 3.4 Angstrom

• Replication.

DNA vs Protein • Both are polymers: Protein : polypeptide; DNA:polynucleotide • Both have sequence specificity • Both have directionality, DNA 5’3’; protein NC Chargaff’s Rules • state that DNA from any cell of all organisms should have a 1:1 ratio (base Pair Rule) of pyrimidine and purine bases and, more specifically, that the amount of guanine is equal to cytosine and the amount of adenine is equal to thymine. This pattern is found in both strands of the DNA • The first rule holds that a double-stranded DNA molecule globally has percentage base pair equality: %A = %T and %G = %C.The rigorous validation of the rule constitutes the basis of Watson-Crick pairs in the DNA double helix.

DNA

DNA

Protein Folding: • A protein with 100 aa will have 100100 different conformations (based on 10 different conformations per aa) – if it switches between conformations in 10-13 s, it will take longer than the life on the universe to explore all the possible conformations. Levinthal Paradox: in vivo, proteins 10-1 – 103 s. So folding is not random, but directed • A protein that has folded in its native state is Unique, Stable, Accessible “USA” Characteristics of the Folded State • Tight packing - compact • Sequence determined / environment modulated • Families and symmetry • Each sequence unique structure • Native state is thermodynamically stable – lowest energy Protein stability and folding • Proteins function depends on its 3D structure • Denaturation: loss of structural integrity and thus function - By: – Heat/Cold, – pH changes, – Organic solvent – Chaotropic agents (create chaos): urea & guanidinium HCl

Denaturation midpoint is defined as that temperature (Tm) or denaturant concentration (Cm) at which both the folded and unfolded states are equally populated at equilibrium.

Disulfide Bridges Disulfide bridges can be disrupted by treating a protein with 2-

mercaptoethanol. The Cys residue is reduced and the Mercaptoethanol is oxidized Anfinsen Experiment in Protein Folding

• Anfinsen wanted to show that the information for protein folding

resided entirely within the amino acid sequence of the protein. He

choose ribonuclease A he treated it with the denaturant urea plus 2ME

to break the disulfide bridges.

• Under those conditions, the protein unfolded. It would refold

spontaneously once he removed urea and 2ME from the folding solution.

Ribonuclease A regained biological activity under those conditions. This

demonstrated that refolding could take place in vitro.

Physics of Folding

• Entropy drives towards the Unfolded states. HB (hydrophobic) interactions exposed

• Enthalpy drives towards the FOLDED stated.

– HB interactions

– H-bonding

– Ionic interactions

• Free energy (G) is the difference. The FOLDED state is more stable.

Steps of Folding

• Unfolded protein bury core hydrophobic a.a. 2 fold molten globule (loose 3 fold) 3 4 (breathing)

• Takes <msec time frame from start to molten globule state.

• Takes up to 1 sec time frame from molten globule state to 3 folded sate.

Most common obstacles to native fold

• Aggregation

• Non-native disulfide bridge formation

• Isomerization of proline (between cis and tran)

Energy funnel for folding

• Multiple folding pathways can occur

• Model this with energy funnel

The funnel explores the energies landscape and conformational space available

• a) No folding intermediates with significant stability

• b) Real life example, multiple stable intermediates before leading to lowest energy form

• c) Only one stable form no intermediate

• d) Many very stable intermediates on every possible path

Protein Folding Follows a distinct path

• 2 structures assemble first then tertiary then quarternary

•

Protein Folding Anfinsen Experiment

Disulfide Bridges Disruption

Physics of Folding

Steps of folding

Energy Funnel

Protein Folding – Distinct Path

Proteostasis : maintenance of cellular protein activity is accomplished by the coordination of may different pathways.

Protein Misfolding – basis for many human diseases

e.g. In time the helices will be cleaved by proteolityc enzymes leaving the beta sheets free to assemble in fibroid structures responsible for Alzheimer disease

PrP = Prion Protein - Cause of many brain disease in humans

The infectious isoform of PrP, known as PrPSc, is able to convert normal PrPC proteins into the infectious isoform by changing their conformation, or shape; this, in turn, alters the way the proteins interconnect. Although the exact 3D structure of PrPSc is not known, it has a higher proportion of β-sheet structure in place of the normal α-helix structure. Aggregations of these abnormal isoforms form highly structured amyloid fibers, which accumulate to form plaques.

Chaperonins / HSP Proteins – HSPs help proteins fold by preventing aggregation

• Recognized only unfolded proteins – Not specific – Recognize exposed HB patches – Prevent aggregation of unfolded or misfolded proteins

• HSP70 – Assembly and disassembly of oligomers – Regulate translocation of ER

• HSP60 (GroEL) & HSP10 (GroES) – Work as a complex

GroEL • Each subunit

– Apical Domain (/ motif) • Opening of chaperone to unfolded protein • Flexible • Hydrophobic

– Intermediate domain ( -helices) • Allow ATP and ADP diffusion • Flexible hinges

– Equatorial domain (-helices) • ATP binding site • Stabilizes double ring structure

– Central cavity is up to 90 Angstroms in diameter • 7 subunits in one ring • 2 rings back to back

GroES • Cap to GroEL • Each subunit

– sheet – hairpin (roof) – Mobile loop (interacts w/GroEL)

• 7 subunits in functional molecule GroEL + GroES work together • GroEL makes up a cylinder

– Each side has 7 identical subunits – Each side can accommodate one unfolded protein

• 1 GroES binds to one side of GroEL at a time – Allosteric inhibition at other side

• Only one side of cylinder is actively folding protein at a time

Protein Stability (thermal) • Protein engineering (mutagenesis) • S-S bridges

– -CH2-S-S-CH2 – Analysis of all possibilities (many) – Energy minimization to reduce to a few plausible candidates – Site selective mutations – Protein synthesis – Assay: e.g.: T4 lysozyme (x-ray structure known); reducing

degrees of freedom (entropy) increases protein stability • Gly & Pro

– Gly freedom – Pro constraints (side chain is fixed by covalent bond to main

chain) – Gly Pro has propensity to increase stability (more delicate) – Gly Ala usually increases stability – Pro Ala usually decreases stability

• Dipolar stability – Helix: N-end (neg a.a.); C-end (+ a.a.) – Increase stability by mutating residues at N-end of helices from

polar to negative (e.g. ASN ASP; SER ASP) • Hydrophobicity in the core (cavity)

– Barnase (bacteria RNAse – 110 a.a.) (struct by NMR & Xray) – Introducing cavities in the core by mutations such as

• Ile Val or Phe Leu • Cavity for a –CH2 leads to stability by 1kcal/mol

– More delicate design – Needs structure

Protein Stability

Proteostasis Protein Misfolding PrP = Prion Protein

GroEL GroES GroEL + GroES work together

B-DNA • Most common; Right handed helices • 10bp/turn; 1 full turn = 34 Angstroms • Width of dsDNA is 20 Angstroms; Helical twist is 36 degrees • B-DNA has distorted helices • Bases within the grooves exposed to solution • Protein binds DNA within these grooves

– Binding of -helix of protein to DNA can bend DNA structure – Major groove binding is most common

How do proteins recognize genes? • Most genes are silent, unless specifically turned on.

– DNA RNA Protein • Switch Protein: Transcription Factor

– Activator – Repressor – Can be regulated by small molecule

• Gene activators and repressors – Main controls of: growth, differentiation, oncogenesis – Recognition: affinity, specificity

Activators & Repressors • DNA (a nucleotide base sequence) is the “lock” and the protein motifs are

the “key”. There are 3 motifs. – DNA: Where?

• Only the edges of the nucleotides are accessible to the solvent or to protein, primarily in the major groove of the DNA double helix

– Protein: How? • Protruding groups from protein surface to contact

nucleotides at base of groove • Motifs

• Motif 1: Helix-turn-Helix – Prokaryotes act as dimer: HTH; Eukaryotes act as monomer:

Homeodomain; Metal free – HTH Motif

• 2 helices connected by loop • 2 motifs for functional binding • 1 helix from each motif interacts with DNA ~ 10bp

– Sequential interaction w/backbone and bp dependent upon specificity

– Interaction of dimer bends the DNA

• For Baterial Helix-Turn-Helix – DNA is distorted; protein is dimeric – H-bonds between sugar-phosphate backbone and protein anchor

DNA to protein – Sequence-specific recognition between DNA bases and -helix – Recognition helices in dimer are separated 34 Angstroms apart,

ie. one turn of B-DNA – If one helix binds to major groove, the second helix binds (34A) to

major groove one turn away. – DNA bends and can interact with other regions of protein. – Examples of Allosteric Effects on Binding

• E.g. Trp Repressor: Small molecule that acts far from protein binding site with DNA causing a conformational change in the recognition helix

• E.g. CAP Binding Protein – catabolite activating protein Activated by cAMP binding; dimer binds major groove; both CBP & DNA are distorted upon binding

• Motif 2: Zinc-stabilized – Helix-turn -sheet – 2 strands + 1 helix coordinated by a zinc ion – 2 Cys + 2 His coordinate the zinc metal – Highly conserved sequence especially around coordination sites – Functional interactions

• 3 fingers wrapping around DNA along major groove • Dimer aligning motifs 34 Angstroms apart and interacting

at major groove – Recognition helix positioning: DNA backbone to

side chain of the loop – Base pair interactions with the recognition helix

side chains • Motif 3: Leucine Zipper

– 2 amphipathic helices form coiled coil to stabilize dimer – Distal, basic region on each helix interacts with major groove of

DNA – Heptad repeats in leucine zipper

• 7 a.a. repeat stabilizes the dimer interactions within the coiled coil

B-DNA How Proteins recognize genes

Helix-Turn-Helix

specificity

affinity

Trp Repressor

CAP

Leucine Zipper

Zinc Finger

DNA-Protein Interactions

Concepts to learn: • Structure of the antibody molecule – IgG • The immunoglobulin fold • Hypervariable loops form the antigen-binding sites • Structure of the major histocompatibility complex

– MHC • The MHC fold and peptide-binding sites • Structure of the T-cell receptor • T-cell receptor recognize antigen ONLY when it is bound in the peptide-

binding site of the MHC Two Types of Immune Systems • Cellular immune system ‐ targets own cells that have been infected ‐ also clears up virus particles and infecting bacteria

‐ key players: Macrophages, killer T cells (Tc), and inflammatory T cells (TH1)

• Humoral “fluid” immune system ‐ targets extracellular pathogens ‐ can also recognize foreign proteins ‐ makes soluble antibodies ‐ keeps “memory” of past infections ‐ key players: B‐lymphocytes and helper T‐cells (TH2) Self vs. Not Self Immune system recognizes, destroys and remembers foreign parasites while

ignoring the host’s cells and proteins • Recognizes foreign molecules = Antigen

– B cells release Immunoglobulins to circulation • T cells have T cell Receptor that recognizes when presented on MHC class 1

protein Antibodies: IgG

Composed of two heavy chains and two light chains • Composed of constant domains and variable domains • Light chains: one constant and one variable domain • Heavy chains: three constant and one variable domain • Variable domains of each chain make up antigen‐ binding site

(two/antibody) • Variable domains contain regions that are hypervariable (specifically

the antigen‐binding site) • Confers high antigen specificity

Immunoglobulin Molecule • Heterotetramer – 2 Light chains +2 Heavy chains – Linked by 4 disulfide bonds – Hinge region of HC gives flexibilbity • Ig fold is distorted barrel

– Each LC contains 2 Ig folds – Each HC contains 4 Ig folds – Distored conformation stabilized by intrachain disulfide

bridges • Antigen recognition at loops between strands at N-terminus of Ig Each IgG recognized only 1 antigen: • Each B cell generates 1 type of Ig that recognizes 1 antigen • HC and LC are different genes • DNA for complete Ig arises from combinatorial joining of exons • Regions at N terminus are most variable

MHC Class 1 & 2:

• Recognition motif is 2 helices

+ 2 antiparallel sheets • Recognition motif connected to TM domain by sandwich • IgG-like domains and • MHC class 1 is monomeric • MHC class 2 is dimeric

Recognition Motif of MHC:

• All TCRs recognize helices of MHC • Foreign peptide lays in cradle between helices • High specificity binding of TCR to foreign peptide within

MHC cradle

Immunoglobulin Fold Constant & Variable Domains Light Chain: V & C Domains IgG antigen recog. at loops

Immunoglobulin G (IgG) Stucture

Immunoglobulin Molecule: IgG Self vs. Not Self

Ea IgG recog only 1 Antigen

Recognition Motif of MHC

MHC Peptide Presentation

MHC proteins present peptides of foreign proteins for T Cell Receptors on T cells

MHC class 1 and 2

TCR Binding to MHC 1

MHC-1 Peptide TCR complex

Enzyme Catalysis-Serine Proteases Concepts to be learned • Activation Energy • Transition State • Example: Proteases • Requirements for proteolysis • Families of proteases • Protein Folds used by proteases for catalysis Catalysis Enzyme: increases rate of chemical reaction, decreases activation energy How? – Binding to the transition state of the substrate (L. Pauling 1946) Reaction Path: Residues of Enzyme Substrate Product

How to Lower G

Enzymes bind transition states best The idea was proposed by Linus Pauling in 1946 Enzyme active sites are complementary to the transition state of the reaction Enzymes bind transition states better than substrates Stronger/additional interactions with the transition state as compared to the

ground state lower the activation barrier Largely ΔH‡ effect Covalent Catalysis A transient covalent bond between the enzyme and the substrate Changes the reaction Pathway -Uncatalyzed: -Catalyzed: Requires a nucleophile on the enzyme –Can be a reactive serine, thiolate, amine, or carboxylate Metal Ion Catalysis Involves a metal ion bound to the enzyme Interacts with substrate to facilitate binding – Stabilizes negative charges Participates in oxidation reactions Serine Proteases Peptide bond cleavage by forming tetrahedral transition states: First Stage: Acylation “Acyl-enzyme intermediate” formed Second Stage: Deacylation “Acyl-enzyme intermediate” is hydrolyzed by water

Serine Proteases: Rx: Peptide Bond Cleavage 4 Requirements 1. Catalytic triad: Ser195, His57, Asp102 Ser forms a covalent bond with substrate specific reaction path His: accepts H+ from Ser, thereby facilitates bond formation, and stabilizes negatively charged transition state Asp-: stabilizes positive charge of His+, increases rate ~10,000 2. Oxyanion binding site: Stabilizes transition state, forms 2 H-bonds to a neg oxygen of the substrate 3. Substrate specificity pocket: Recognition/identity (trypsin;chymotrypsin) 4. Non-specific binding site for polypeptide substrates Chymotrypsin Mechanism 1. Substrate binding 2. Nucleophilic attack 3. Substrate cleavage 4. Water comes in 5. Water attacks 6. Break-off from enzyme 7. Product dissociates Chymotrypsin Structure 2 domains Each domain: antiparalled -barrel, six -strands 4 (1-4) 2 (5,6) Greek Key Motif, beta hairpin Active Site: 2 loop regions from each domain Substrate specificity pocket- Aromatics – Trypsin: R or K – Elastase: Pocket blocked small uncharged Bacterial Subtilisin : , type (J. Kraut, UCSD) 4 a-helices surrounding 5 parallel B-strands Active site: -C-end of the central B-strands -Catalytic triad: S,H, D Carboxypeptidase: (catalysis by induced electronic strain on substrate) Zn2+ Protease -Glu 270 directly attacks the carbonyl carbon of the scissle bond to form a “covalent mixed-anhydride intermediate” -Zn2+ binding polarizes the carbonyl -“environment”, non-polar, induced dipole -Facilitates hydrolysis by water

Enzymes accelerate chemical reactions by decreasing the activation energy

a.a. in general acid/base catalysis: Hydrolysis of peptide bonds

Chymotrypsin uses most of the enzymatic mechanisms

Chymotrypsin Two anti-parallel domains

Acylation and Deacylation of the Acyl-Enzyme Intermediate

Tetrahedral Transition State Active site of chymotrypsin with substrate (see mechanism, next slide)

Specificity pocket

Subtilisin Active site of subtilisin

The Lipid Bilayer • Fundamental structure of cell membranes • Of the same rank of importance as the DNA double helix and the a-helix and

B-sheets of proteins • A thin film (40 Angstoms thick) with a hydrophobic core ( ~2) separating two

aqueous compartments (~80) QUESTIONS • How membrane proteins fold? • How charged and polar molecules go through lipid bilayers? Membrane Proteins: Concepts to learn today • The interplay between the bilayer hydrophobic – interfacial environment and

the protein which dictate the membrane protein fold • How membrane proteins provide a solution to the question of how charged

and polar molecules traverse bilayers Micelles: favored when head group cross section area is larger that acyl side

chain for ex. single side chains FA such as SDS • When this type of molecule is added to water, the non-polar tails of the

molecules clump into the center of a ball like structure, called a micelle, because they are hydrophobic or "water hating". The polar head of the molecule presents itself for interaction with the water molecules on the outside of the micelle.

• Lipids with head cross section= to head form lipid bilayers. Structure of Membrane Lipids Membrane lipids are a group of compounds (structurally similar to fats and oils)

which form the double-layered surface of all cells. membrane lipids have polarity with one end that is soluble in water ('polar') and an ending that is soluble in fat ('nonpolar'). By forming a double layer with the polar ends pointing outwards and the nonpolar ends pointing inwards membrane lipids can form a 'lipid bilayer' which keeps the watery interior of the cell separate from the watery exterior.

Spingomyelin

• Most phospholipids contain a diglyceride,(A diglyceride, or a diacylglycerol (DAG), is a glyceride consisting of two fatty acid chains covalently bonded to a glycerol) a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from sphingosine instead of glycerol.

• Archaeal lipids lack the fatty acids found in Bacteria and Eukaryotes and instead have side chains composed of repeating units of isoprene.

Lipid Bilayer in Aqueous Solution: Lipid bilayers in water are unstable: they tend to close on themselves forming a more stable vesicle (it eliminates exposed hydrophobic regions)

Membrane Proteins: Membrane proteins responsible for permeability: a b and c are transmembrane integral proteins, they span the lipid bilayer. E and d are peripheral membrane protein, they bind integral membrane protein.

Bacteriorhodopsin: 7 hydrophobic helices crossing the membrane, 7 20 aa (enough to span the membrane)sequences segments can be identified. Helix bundle.

Porins: Beta barrel

Beta barrel sequence(Porin):Recall that side chain are arranged alternating up and down so every other aa is hydrophobic (all the ones on one side are in contact with the membrane) the other may or may not be hydrophobic.

Polarity Scale: Calculate the free energy of the transmembrane helix . When an aa is moved from an hydrophobic environment to water, the free energy change is measured(hydropathy index). Polar aa are very exergonic(loss of energy)b/c the form bonds, and non polar are the opposite.

Phe = 15.5 kcal/mol = very stable; Arg = -51 kcal/mol = very unstable. Lateral & Transverse Diffusion: Flippases catalyze transverse diffusion making it faster. Membranes & Temperature As Temperature increases membranes go from solid or crystalline structure to fluid. Long chain FA favor the solid state while unsaturated FA favor the liquid form Aromatic Belt: …is located at the interface between the lipid bilayer and water. Trp and Tyr are aromatic aa that can be both hydrophobic and polar(charged). It serve to anchor the protein and stabilize it in the membrane. Liquid Ordered vs. Disordered State: Long chain saturated FA favor the Lo state(gel), while unsaturated and short chain FA favor the liquid state. Cholesterol: • As a buffer: Cholesterol also affect membranes: it interacts with unsaturated lipids making the more rigid. While, when associated to sphingolipids and saturated lipids, it makes them more fluid. • Cholesterol tends to associate with sphingolipids to form regions in Lo state surrounded by cholesterol poor regions in the Ld state. These regions are known as RAFT. Calculation

Glycophorin presence of sugars (shown in green diamond objects) This is 131 a.a. long.

Outside to inside the bilayer is about 40 A thick; there are about 20a.a. that are hydrophobic; 20 a.a. / 3.6 a.a. (which is the # of a.a. per one turn of the alpha helix) = 5.6 turns

Saturated Lipids

Un-saturated Lipids

Lipid bilayers in aqueous solution

Membranes are Permeable

As permeability increases, larger molecules pass through

Membrane Proteins

Bacteriorhodopsin:

Porins:

Beta barrel sequence(Porin):

Polarity Scale Hydropathy Plot

Lateral & Transverse Diffusion

Membranes & Temperature

Aromatic Belt

Liq

uid

Ord

ered

vs.

D

iso

rder

ed S

tate

:

Cholesterol Calculation

Bacteriorhodopsin: 7 transmembrane -helices

• Binds Retinaldehyde via a Schiff Base, a covalent bond between K216 and retinal.

• Molecular switch which transforms light energy into a H+ electrochemical gradient, i.e., a H+pump

• It catalyzes vectorial H+ transport by a H+-relay mechanism: A H+ from the Schiff base is donated to Asp85 while a H+ from Asp 96 reprotonates the Schiff base

Bacteriorhodopsin is a protein used by Archaea, the most notable one being Halobacteria. It acts as a proton pump; that is, it captures light energy and uses it to move protons across the membrane out of the cell.[1] The resulting proton gradient is subsequently converted into chemical energy

Schiff Base, named after Hugo Schiff, is a compound with a functional group that contains a carbon-nitrogen double bond with the nitrogen atom connected to an aryl or alkyl group, not hydrogen.[1] Schiff bases in a broad sense have the general formula R1R2C=NR3, where R is an organic side chain.

Retinal (trans to cis) Bacteriorhodopsin exists in 2 states: Tense (T) – binds trans-retinal Relaxed (R) – binds cis-retinal T state: Schiff Base is protonated Excitation by photon Isomerization H+

transferred from Schiff base to Asp 85 R state: H+ transferred from Asp 96 to Schiff base and H+ from Asp 85 is transferred to Extracellular Space H+ transferred from Cytoplasm to Asp 96 Bacteriorhodopsin Photocycle

• T state: Schiff Base is initially protonated - Excitation by photon - photoIsomerization - H+ transferred from Schiff base to Asp 85

• R state: H+ transferred from Asp 96 to Schiff base and H+ from Asp 85 is transferred to Extracellular Space- H+ transferred from Cytoplasm to Asp 96.

Porin: • Homotrimeric protein. 3 monomers form this protein • Each monomer is made of a 16 stranded Up/Down β-Barrel. • Subunits interact by polar loop and hydrophobic side chain interactions. (The

side surface is very hydrophobic b/c that’s the part in the lipid bilayer which is also very hydrophobic.)

• Periplasmic End: short and smooth • Cytoplasmic End: funnel shaped, made of long loops with hydrophilic residues. • Eyelet = loop connecting β5 and β6 extending into the central open cavity of the

barrel. • Cavity is lined with charged residues, arranged in an electrochemical gradient. Porin Aromatic Belt - mainly Phe and Tyr • Has polar and hydrophobic character to interactive with the hydrophilic space of

the membrane extracellular solution and hydrophobic internal space of the membrane.

• Helps stabilize protein in membrane.

Transporters: There are two classes of transporters. Antiporter: e.g. Na/K ATPase Symporter: e.g. Na+/Glucose; concentrate on the Na+ electrochemical gradient. Create an electrochemical gradient with Na/K via energy of ATP

Schiff Base

Bacteriorhodopsin

Retinal trans vs cis

Bacteriorhodopsin Photocycle

Porin Trimer

Porin Aromatic Belt

K+ Channel • Homotetrameric protein. • Ion pore made between the four monomers. • Each monomer is made of 2 transmembrane alpha helices, 1 pore helix and

a cytosolic tail. • Selectivity Filter – formed from the backbone carbonyl Oxygens from the

loops connecting pore helix to the inner transmembrane helix. • The structure is stabilized by the packing of residues from the loop into

the pore helix. Backbone Carbonyls: What are backbone carbonyls? Look at the white loop on

the left side. Blown up view on the right. Two K+ ions with Gly, Tyr, Gly, Val, Thr lined along the side. These a.a. stabilize the K+ ion. There are 4 subunits. Each subunit contributes these a.a. When the 4 subunits come together, that’s what forms the pore. The K+ ions are bonded or liganded or stably bonded to the protein via these a.a. The bottom K+ ion is coordinated by EIGHT (8) oxygens. Not only the 4 shown here. Each of the 4 subunits contributes 2 oxygens.

K+ Channel Selectivity How is the channel selective for K+? The protein acts as surrogate Water

• K (OH2)8+ = hydration shell (8 H2O needed to stabilize K+)

Within the K+ Ion Channel only: Resolvation Energy ~ Desolvation Energy

• Na (OH2)6+ = hydration shell (6 H2O needed to stabilize Na+)

• Within the K+ Ion Channel only:

• Resolvation Energy <<< Desolvation Energy

• Rapid ion flow through the selectivity filter is facilitated by ion-ion repuslion, with one ion pushing the next ion through the channel

Electrostatic Repulsion **When two K+ occupy adjacent sites, electrostatic repulsion forces them apart. Thus, as a K+ enters the selectivity filter from one side, repulsive force from the next K+ entering the filter pushes the first ion through the pore and out the other side, namely out of the cell. Modular Design: Eukaryotic Kv channels are by modular design Conservation: Sequence of the voltage sensor is conserved in various types of

voltage gated K+ channels in the prokaryote.

Nicotinic Acetylcholine Receptor (nAChR) • found in muscle; in the brain it’s the alpha7 AchR • ion channel for influx of Na+, Ca++, K+ • the protein is a PENTAMER and ligand-gated • gate opened by ACh binding • altogether this protein has 20 transmembrane segments • (4 TM alpha helices/subunit x 5 subunits = 20 TM segments) • ONLY ONE of the four a-helices, the M2 segment, forms the center of

the pore. The five M2 segments come together to form the pore of the channel. The M2 helix has polar a.a. that line the lumen of the channel which allows ions to go through.

• So how does the channel open? When ACh binds, a conformational change occurs. As the M2 helices twist slightly, the five Leu residues rotate away from the channel and are replaced by smaller polar residues. This gating mechanism opens the channel, allowing passage of Ca++, Na+ or K+

TTX K+ Channel Tetramer K+ Channel Tetramer, side view

K+ Channel: Pore helix & filter

Backbone Carbonyls

Electrostatic Repulsion

K+ Channel Selectivity

Modular Design

Conservation

Photosynthesis

Summary: • The reduced cofactors pass electrons into the electron transport chain in

mitochondria • Energy of sunlight creates charge separation in the photosynthetic rxn complex • Stepwise e- trans is accopanied by the directional transport of protons across the

mbn against their [] gradient • Energy in electrochem proton gradient drives synthesis of ATP by coupling flow of

protons via ATP synthase to conformational changes that favor formation of ATP in the active site.

Photosynthesis and Membranes • The proteins that participate in the light reactions of photosynthesis are located in

the thylakoid membranes of chloroplasts and in the cell membrane of bacteria. • Photosynthesis transforms light energy into electrochemical energy • Light reactions result in:

– The formation of reducing power – The generation of a transmembrane proton gradient for the synthesis of

ATP, and – The production of O2

Light induced charge separation • Ground state to excited state. • Change in energy brought about by absorption of a photon. • This creates a charge separation. • Donor and acceptor have opposite charges • Mbn is 30A thick. • Charge is separated by bilayer. • You get a net separation of charge. Photosynthetic Pigments a. Bacteriochlorophyll b. Ubiquinone c. Carotenoids Energy transfer Occurs from accessory pigments to the reaction centers Why are these pigments involved? Green areas = pigments which are antennae for the photons of light Blue = rxn cneter whre the primary rxn occurs. Bacterial PS Rxn Center What is the Photosynthetic Reaction Center? This is a cartoon of the protein. 3 subunits: Yellow = L = 5 TM helices (low molecular weight) Red = M = 5 TM helices (medium molecular weight) Green = H = 1 TM helix (high molecular weight) Blue = cytochrome (it is NOT in the membrane) Black = pigments With gentle treatment we can remove the cytochrome from the reaction center. Note: M-subunit interacts with cytochrome even though cytochrome is not a membrane protein.

PS Rxn Center with Pigments: • This is the cytochrome with the heme group. • The ion can be reduced or oxidized. • Then enter into the membrane where e- is transferred to chlorophyll • Have a special pair of chlorphylls; 2 accessory chlorophylls; 2 pheophytins; 2

quinones;1 iron • What starts this reaction? A photon of light. Pigments in Membrane: are bound to transmembrane a-helices of L and M

subunits Hydropathy Plots Hydropathy plots of M-subunit and L-subunit have 5 potential TM segments a.a. sequence Red = charged residues Blue = poloar residues Yellow = glycine Green = hydrophobic residues Light-harvesting Complex • LH2 • LH1 Arrangement of Chlorophyll: Relative arrangement of chlorophylls in the

membrane with respect to LH1, LH2 and the reaction center • Side / cross-sectional view of 1 subunit of each of LH2 and LH1. • There are many LH2 units, smaller • There’s only one LH1 unit, and it’s large. LH1 houses the PRC • How is light energy transferred from complex to complex? b/c of proximity. The complexes are very tightly packed. • Hypothesis of how the system works: in the membrane the two light

complexes are tightly packed.875nm and 800nm == wavelength at which they absorb light.

Energy Transfer Energy transfer from accessory pigments to the reaction centers Structure of Photosystem II, in chloroplasts, in plants. • Pretty much the same system as in baceteria but this is in

plants/chloroplasts. • Why important? • Very efficient (as measured by input and output) • Ideal system where 1 photon 1 equivalent of energy • The PS: 1 photon 0.92 equivalents of energy (almost 1:1) • Have not been able to replicate this level of efficiency synthetically.

The light reactions of photosynthesis Structure of a chloroplast Light induced charge separation

Photosynthetic Pigments Energy transfer

Bacterial Photosynthetic Rxn Cntr Photosynthetic Rxn Cntr w/Pigments

Pigments in Mbn

Hydropathy Plots a.a. sequence

LH2 LH1 LH2

LH2

Arrangement of Chlorophylls Energy transfer

Structure of PS II

Signal Transduction Signal outside cell is recognized Transmission across membrane Effect inside cell Signal Transducing Systems – 5 features Specificity, Amplification, Modularity, Desensitization/Adaptation, Integration General Overview of Signal Transduction & Molecular Circuits • primary messenger (hormone-H) binds to the extracellular part of a

membrane-embedded receptor (R) • The HR complex generates a second messenger inside the cell which

activates proteins that alter the biochemical circuitry inside the cell • Then, mechanisms are activated to terminate the signal transduction

pathway Membrane embedded receptor Proteins transmit information into the cell • G-Protein Coupled Receptors: (a) The Ras protein (small G-protein), (b) like

the G subunit of heterotrimeric G proteins (large G-protein), cycles between an inactive GDP-bound form and active form bound to GTP

• Tyrosine kinase modules of some receptors upon dimerization are activated by cross- phosphorylation. Phosphorylated tyrosines serve as docking sites for adaptor and signaling proteins which permit further propagation of the signal.

• Net result is amplification, fidelity and diversity Structure of G heterodimer G = 7 four-stranded beta sheets that form a propellar G = an alpha helix that’s shown in yellow 6 General Types of Signal Transducers • G-protein Coupled Receptors that indirectly activate (through GTP-binding

proteins, or G-proteins) enzymes that generate IC 2nd messengers. E.g. -adrenergic Receptor system that detects adrenaline/epinepherine. E.g. Ras

• Receptor Tyrosine Kinases – plasma mbn rec that are also enzymes. When one of these receptors is activated by its EC ligand, it catalyzes the phosphorylation of several cytosolic or PM proteins. E.g. Insulin Receptor E.g. Growth Hormone & Epidermal Growth Factor Receptor (EGFR)

• Receptor Guanylyl Cyclases – IC 2nd messenger for these is cGMP • Gated ion channels

E.g. Voltage-gated K+ channels E.g. nAChR

• Adhesion receptors • Nuclear Receptors

Biological Functions mediated by 7TM Receptors What can G-proteins do? Common 2nd Messengers: cAMP, cGMP, Ca++, IP3, DAG Adrenaline = Epinepherine: the fight or flight hormone •Hormone made in adrenal glands •Mediates stress response: mobilization of energy •Binding to receptor in muscle or liver cell induces breakdown of glycogen •Binding to Receptor in adipose cells induces lipid glycolysis •Binding to receptor in heart cell induces increase in heart rate GPCRs – The Receptors G-Protein Cycle GTP = active form; GDP = inactive form Activated -subunit hydrolyzes GTP to GDP Special proteins help GTP hydrolysis (regulators of G-protein signaling , RGS) and units reassemble into inactive form. Why use G-Proteins? • Amplification at two steps: – Active receptor can activate multiple G-proteins (by causing GTP to

replace GDP) – Each downstream target (e.g. adenylate cyclase) can produce many 2nd

messengers (e.g. cAMP) • Cells can regulate how long the switch is turned on: – Different RGS molecules in different cells or at different times.

Activated G-protein Receptor

Trimeric molecular amplifier (,, subunits; and are lipid-linked thus membrane bound

Bind to 7TM receptor on the cytosolic side (as rep by red 7TM helices)

Receptor is activated catalyze exchange of GTP for GDP on trimer dissociation of from /

The separated G-GTP and G molecules activate various effector molecules

NOTE: the receptor is embedded in the membrane. BUT, the subunits are attached to the membrane by lipid anchors

G-protein activatin of adenylate cyclase by horomone binding

Hormone on the extracellular side binds to receptor such as B-adrenergic R and activates a G-protein on the cytoplasmic side.

Activated G-protein (-subunit with bound GTP) dissociates from and subunits and activates adenylate cyclase to produce cAMP which is a 2nd messenger that affects a diverse range of cell processes.

The Adrenergic Receptor (or adrenoceptors) are a class of G protein-coupled receptors that are targets of the catecholamines, especially norepinephrine (noradrenaline) and epinephrine (adrenaline).

Signal Transduction Five features of signal transducing systems

Structure of G heterodimer

6 General Types of Signal Transducers

Common 2nd Messengers

Signal Amplification

GPCRs – the Receptors

The Adrenergic Rec.

Activation of G-Protein Receptor

G-protein Activation of Adenylate Cyclase (3 cartoons, same example)

A heteromeric G-protein

G-protein

G-Protein Coupled Receptors

The Basic Structure – Monomeric G-protein Ras

The Basic Structure – Heteromeric G-protein

G-protein Cycle

Self-inactivation of G-proteins

RAS The Odd Case of Ras • Ras lacks the Arg that stabilizes the negative charge on the phosphate! • Ras is very very very slow at hydrolyzing GTP – is this why? • Ras GTPase Activating Protein (GAP) supplies the necessary Arg residue and

speeds up the reaction nearly 100,000 fold. Why is all this important? • Ras is a signalling molecular activated by various pathways, including cell

growth signals • Ras is mutated in 25% of humor tumors

• Mutations inactivate the GTP hydrolysis step, thereby keeping Ras swtiched “on” – and thus Ras keeps on stimulating cells to divide

CHOLERA TOXIN CT is an AB toxin B is a pore that allows toxin entry into the cells A is an enzyme that stabilizes the active GTP-bound form of G Active G activates Protein Kinase A (PKA) PKA activates (by phosphorylation) a Cl- channel and a Na+/H+ exchanger The net consequence is massive loss of NaCl and water from the intestine.

How Ras gets activated

Enzyme-linked Membrane Receptors • Many membrane receptors consist of:

– Extracellular ligand-binding domain, and of – Intracellular catalytic domain

• The most common catalytic domains have tyrosine kinase activity – Adds a phosphate group to itself; auto-phosphorylation leads to a

conformational change allowing binding and catalytic phosphorylation of specific target proteins

– Adds a phosphate group to a tyrosine in specific target proteins • Some catalytic domains have guanylyl cyclase activity

– Convert GTP to cGMP, a secondary messenger Tyrosine kinase modules of some receptors upon dimerization are activated by cross- phosphorylation. Phosphorylated tyrosines serve as docking sites for adaptor and signaling proteins which permit further propagation of the signal. Growth Hormone – e.g. of Rec Tyr Kin signaling EC domain has 2 subunits with GH attached in blue GH Protein Is a 4 helical bundle with up-up-down-down configuration. Structure of 1 Subunit of GH: No alpha helices GH Receptor Dimerization Note the surface of GH on right side presented to the subunit compared to the left. Surface of GH on left side is larger, longer, more widespread. The stable complex leads to dimerization. It’s really the first binding on the left side that affects the second binding. GH Receptor Dimerization – Cartoon View •The receptors are independent. •The critical event is the binding of GH to the left subunit which then brings the 2nd subunit together. •This is called CROSS PHOSPHORYLATION; aided by adaptor proteins which are noncatalytic proteins that hold together other protein molecules.

CROSS TALK:

Where the Beta adrenergic Rec “talks” to the Insulin receptor Modularity & Conservation EC domains are different IC domains all have tyrosine kinases

Insulin – the hormone for glucose uptake and metabolism • Insulin is a peptide hormone that is produced by the -cells of islets of Langerhans in

the pancreas • Insulin is produced and released from the pancreas in response to nutrients such as

glucose • Insulin reaches target cells, such as liver, muscle, or fat tissue cells via bloodstream • Binding of insulin to the insulin receptor initiates a cascade of events that leads to

increase glucose uptake and metabolism • Inability to make or sense insulin diabetes Insulin Signaling Cascade – Ligand Binding • Insulin binding to the extracellular domains of the receptor activates the catalytic

domain inside the cell • Catalytic domain in one receptor phosphorylates Tyr residues in another receptor • Receptor auto-phosphorylation allows binding and phosphorylation of protein IRS-1 NOTE: 3 Tyrosines are phosphorylated “Triply” What happens once tyrosine is phosphorylated? SH2 Domain: Alpha-beta motif • Structure of SH domain = adaptor protein • Stretch of protein on beta part is a phospho tyrosine 1 • When phosphotyrosine bound the surface changes • SH2 domains bind proteins with phosphotyrosine residue

SH2 domain structure.

• SH2 domains allow proteins containing those domains to dock to phosphorylated

tyrosine residues on other proteins. SH2 domains are commonly found in adapter

proteins that aid in the signal transduction of receptor tyrosine kinase pathways.

• it contains 2 alpha helices and 7 beta strands. Research has shown that it has a high

affinity to phosphorylated tyrosine residues and it is known to identify a sequence

of 3-6 amino acids within a peptide motif

• SH2 bind pTyr-Glu-Glu-Ile. • SH3-binding epitopes of proteins have a consensus sequence that can be

represented as a regular expression or Short linear motif: -X-P-p-X-P- 1 2 3 4 5 • with 1 and 4 being aliphatic amino acids, 2 and 5 always and 3 sometimes being

proline. The sequence binds to the hydrophobic pocket of the SH3 domain

SH2 & SH3 Domain Binding Conditions

(a) Conditions for binding to SH2 domain: proximal presence of 2 negatively charged side chains e.g. glutamate

(b) Conditions for binding to SH3 domain: proline rich helix, where the helix has 3 a.a. per turn.

SH3 Domain Structure

• Proline-proline fold.

• SH3 domain = beta barrel

Receptor Tyrosine Kinases e.g. Growth Hormone

Signaling Pathways: G-Protein & RecTyrKin

GH Rec with GH bound

Growth Hormone Protein

Structure of 1 subunit of GH Rec

GH Rec Dimerization - GH Rec with GH bound

GH Rec Dimerization – Cartoon View

GH Rec Dimerization – “Cross Phosphorylation” via Adaptor Proteins

Crosstalk between a Tyrosine Kinase Receptor & a GPCR

Insulin Signaling Cascade Receptor Tyrosine Kinases e.g. Insulin

Modular structure of signaling proteins

Modularity & Conservation

SH2 Domain Structure SH2 & SH3 Domain Binding Conditions

SH3 Domain Structure

Insulin Signaling Cascade – Ver 2