Membrane Protein Function and Cellular Signalling

Membrane Protein Function

• Structural changes facilitate function.

Learning Objectives

• Explain how membrane protein structure is crucial for ion/molecule movement and signal transmission.
• Describe essential features of the main types of signaling pathways.
• Identify the roles of lipids and enzymes in transmitting signals across the membrane and throughout the cell.
• Differentiate between passive, primary active, and secondary active transport.
• Review the importance of binding for protein function and how binding affinity is defined and assessed.

5HT2A Serotonin Receptor

• Drug/hormone binding is important.
• Cholesterol is present.
• The slide shows (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate, which is similar to cholesterol but found inside the cell membrane.
• The question posed is, what happens after binding?

Cellular Signalling

• All physiological processes involve biochemical interactions and reactions, allowing a cell to carry out its function and adapt.
• The binding of signaling molecules (ions, hormones, sugars) to their receptors initiates processes such as metabolic pathways and gene expression.
• Proteins are essential for carrying out this response but must be regulated.

Signal Transduction Cascades

• Common components:
  • Specific binding to a signaling molecule.
  • Reception of the message by a receptor (usually an integral membrane protein).
  • Relay of the primary message via an intracellular secondary messenger.
  • Amplification and transduction of the signal.
  • Response and termination of the signal cascade.

G-Protein-Coupled Receptors (GPCRs)

• GPCRs contain 7 transmembrane (TM) segments.
• They are part of a superfamily of membrane proteins.
• Conformational changes release the G proteins.
• Can bind many different ligands (natural and synthetic).
  • Natural: serotonin, epinephrine, prostaglandins, dopamine, psilocin/psilocybin
  • Synthetic: morphine, histamine, LSD
• Binding is key for GPCR specificity.

Characterizing Binding Interactions

• Non-covalent interactions (ionic bonds, hydrogen bonds, van der Waals interactions) between amino acid side chains and the molecule’s functional groups influence binding affinity.
• Binding affinities characterize non-covalent interactions between biomolecules (proteins, ligands, cofactors, substrates, drugs, etc.).
• $K<em>d$ values are dissociation constants; a lower $K</em>d$ indicates stronger binding.
• Binding is saturable based on stoichiometry and reversible for non-covalent interactions.
• Kd = [A][B] / [AB] = k{off} / k{on} = Ka^{-1}
• A + B [k{on}][k{off}] AB

β2-Adrenergic Receptor

• Brian Kobilka and co-workers solved the structures of the β2-adrenergic receptor in inactive and active states.
• Required 20 years of protein engineering.
• Ligand binding induces small changes in TM5 on the extracellular side.
• A 14 Å movement in TM6 transmits the signal inside.
• Major conformational changes in TM6 promote Gα activation.
• Kobilka shared the 2012 Nobel Prize in Chemistry for his work on GPCRs.

Epinephrine Binding

• Hormone signaling on the extracellular face induces a conformational change, releasing the Gα subunit in the GTP-bound state.
• Activation of adenylyl cyclase produces cAMP (a secondary messenger).
• cAMP can activate other enzymes, including Protein Kinase A (PKA), a transferase that can phosphorylate and activate/inactivate other enzymes.
• Regulation of the cascade can be mediated by post-translational modifications, disrupting binding interactions, metabolizing molecules, or through protein degradation.

Termination of Epinephrine Signalling

• The most effective way to turn off epinephrine signaling is:
  • To compete epinephrine.
  • ATP.
  • R & GDP. Once epinephrine leaves, IMG is going back.

Ras Proteins

• Members of the superfamily of small GTPases that bind and hydrolyze GTP.
• Activated in numerous signaling pathways that initiate cell proliferation and apoptosis.
• A conformational change is seen in the switch I and switch II motifs upon phosphate release (GTP → GDP).
• Defects in GTP hydrolysis can lead to uncontrolled signalling and cancer.

Signalling and Human Health

• Defects at any point along the pathway can lead to disease.
• Post-translational modifications and conformational changes play a key role in these pathways.
• Mutations in the receptors or effector proteins can prevent ligand-receptor interactions or the protein-protein interactions needed for activation/inactivation.
• Knowing the structure of (membrane) proteins is important to understand their function.
• Drugs can be designed to bind and inhibit or stimulate the proteins involved in signalling to modulate the cellular response.

Other Types of Signalling

• Enzyme-linked Receptors:
  • Usually contain a single transmembrane segment that may be homodimers or dimerize upon ligand binding.
  • Activation leads to auto-phosphorylation or phosphorylation by tyrosine kinases.
  • Examples: insulin, epidermal growth factor (EGF), Jak/STAT.
• Phospholipid mediated Signalling:
  • Phospholipases hydrolyze phospholipids to produce other 2nd messengers like diacylglycerol (DAG) or IP3 leading to the release of calcium from the ER.
  • Examples: Eicosanoid and AKT signalling.

Hormone Competition

• Insulin and epinephrine are competing hormones.
• Phosphorylation of the Insulin Receptor Substrate (IRS)-1 and activation of the pathway also leads to phosphorylation of the β-adrenergic receptor by Protein Kinase B (PKB).
• This post-translational modification leads to internalization and degradation, terminating GPCR signalling.

Membrane Transport

• Small, uncharged or lipophilic molecules may cross by passive diffusion (slow & concentration dependent).
• Transport is essential for life:
  • Nutrients in - garbage out.
  • Inorganic ions in and out.
• Integral membrane proteins are important for transport via:
  • Facilitated diffusion.
  • Active transport with/without ATP.

Molecule Permeability

Sort the following molecules based on their permeability across the membrane from most to least permeable:

1. Oxygen
2. Water
3. Protons
4. Alanine

Facilitated Diffusion

• Facilitated transport depends on binding sites on membrane proteins.
• The rate of transport (v) is saturable at high substrate concentration (all binding sites are occupied).
• A hyperbolic curve is similar to simple catalytic enzymes.

Channel Proteins

• Membrane transporters that facilitate diffusion are also known as (ion) channel proteins.
• The structure of the membrane protein is key for its function.
• Important features of ion channels:
  • Selectivity (K+ vs. Cl- or K+ vs. Na+).
  • Rapid conductance of ions ($10^8$ / sec).
  • Can be gated (open/closed) due to stimuli.

Potassium Ion Channel

• Essential for many cellular processes:
  • Regulation of cell volume
  • Secretion of hormones
  • Electrical impulse formation (esp. neurons)
• Each subunit contributes a selectivity filter of 5 amino acids (TVGYG) that contribute to K+ binding.
• 4 backbone carbonyls and the Thr side-chain hydroxyl bind the K+ ions.
• Changing the sequence alters the selectivity for other cations.

Gating the Potassium Channel

• In response to specific stimuli (voltage gating/intracellular pH change), helix bending at a conserved Gly residue occurs in the regulatory domain.
• Gly99 acts as a molecular hinge to open/close (gate) the channel.

Sodium-Potassium Pump

• Pumps K+ in.

Beta Barrel Proteins

• Integral membrane proteins may also be composed of β strands that form a pore in the membrane (e.g., porins).
• The amino acids facing the inside of the pore are hydrophilic, while those on the opposite side of the β strands are hydrophobic.
• Beta strands are more extended, requiring fewer amino acids to span the bilayer.
• Less selective.

Beta Strand Design

• Alternating Hydrophobic vs Hydrophilic amino acids are needed.
• Example: TvI ~ Tv I (alternating Hydrophobic/Hydrophilic).

Active Transport

• Active transport is the movement of molecules against their concentration gradient.
• In primary active transport, the breakdown of ATP, light energy, or the passing of electrons generates energy for transport.
• Secondary transporters use the gradient of one molecule to power the formation of another (e.g., Na+-glucose transporters).

Conformational Change in a Flippase

• MsbA – a bacterial lipid transporter that moves phospholipids into the cell.

Bacteriorhodopsin

• Found in Halobacterium salinarum in concentrated purple patches in the membrane (75% bR protein:25% lipid).
• A retinal cofactor contributes to the purple colour of the protein.
• Light energy induces conformational changes in the cofactor, allowing the protein to move protons out of the cell.
• This generates a proton gradient that is used to make ATP by ATP synthase for other reactions.

Bacteriorhodopsin Structure

• A 7TM protein with a retinal prosthetic group covalently attached to Lys216 via a Schiff base.
• The absorbance of light induces a conformational change from all-trans to 13-cis-retinal, altering the pKa values of functional groups for proton transport.

Proton Hopping

• Individual protons are not directly transported/pumped across the membrane but rather passed from one functional group to another.
• As one hydronium ion gives up a proton, a water molecule some distance away acquires one, becoming a hydronium ion.
• Proton ‘hopping’ is much faster than true diffusion and explains the remarkably high ionic mobility of H+ ions.

Key Messages

• Conformational change is key for transport and signaling and is essential for the regulation of activity of membrane proteins and downstream signalling enzymes.
• Non-steroidal receptors are integral membrane proteins with a variety of structures (single TM vs. multi-pass), resulting in the creation of secondary messengers for protein activation/inactivation in the cell.
• Transport can be general or specific based on the structure of the membrane protein and is either concentration dependent or based on the availability of ATP or a co-transporter.