Membrane Protein Function and Signaling

Membrane Protein Function

Learning Objectives

• Explain how the structure of a membrane protein is crucial for its ability to move ions, molecules, or transmit signals across the membrane.
• Describe the essential features of the main types of signaling pathways.
• Identify the role lipids and enzymes play in transmitting signals across the membrane and throughout the cell.
• Distinguish between passive and primary vs. secondary active transport.
• Review the importance of binding for protein function and how binding affinity may be defined and assessed.

Cellular Signaling

• 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

• Signal transduction cascades have many components in common:
  • Binding specifically to a signaling molecule in response to a physiological stimulus.
  • Reception of the message by the receptor, usually an integral membrane protein.
  • Relay of the primary message to the cell interior by the generation of an intracellular secondary messenger.
  • Amplification and transduction of the signal.
  • Response then termination of the signal cascade.

G-Protein-Coupled Receptors (GPCRs)

• GPCRs contain 7 transmembrane (TM) segments.
• They are part of a super-family of membrane proteins.
• Conformational changes release the G proteins.
• Can bind many different ligands:
  • 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 the amino acid side chains and molecule’s functional groups influence the binding affinity.
• Binding affinities can be used to characterize and compare the non-covalent interactions between two biomolecules (proteins, ligands, cofactors, substrates, drugs, etc.).
• Kd values are dissociation constants; a lower Kd equals stronger binding.
• Remember, binding is saturable based on stoichiometry and reversible for non-covalent interactions.
• Kd = \frac{[A][B]}{[AB]} = \frac{k{off}}{k{on}} = Ka^{-1}
• A + B \rightleftharpoons AB

β2-Adrenergic Receptor

• Brian Kobilka and co-workers solved the structure of the receptor in the inactive and active states.
• 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.

GPCR Signaling

• 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 is important and can be mediated by post-translational modifications, disrupting binding interactions, metabolizing molecules, or through protein degradation.
• Turning off epinephrine signaling:
  • Competition with epinephrine.
  • R & GDP
  • Once epinephrine leaves, IMG goes 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 can be seen in the switch I and switch II motifs upon phosphate release (GTP → GDP).
• Defects in GTP hydrolysis can lead to uncontrolled signaling and cancer.

Signaling 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 also be designed to bind and inhibit or stimulate the proteins involved in signaling to modulate the cellular response.

Two Other Important Types of Signaling

• 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 Signaling:
  • 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 signaling.

Hormone vs. Hormone – who will win?!?

• 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 signaling.

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.

Permeability Across the Membrane

• From most to least permeable:
  1. Oxygen
  2. Water
  3. Protons
  4. Alanine

Facilitated Diffusion is Saturable

• Facilitated transport is dependent on the presence of binding sites on membrane proteins.
• The rate of transport (v) is saturable at high substrate concentration (i.e., all binding sites are occupied).
• A hyperbolic curve is similar to what is seen for 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.

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, and you need less amino acids to span the bilayer.

Designing a Beta Strand

• Alternate hydrophobic and hydrophilic amino acids

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 & 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 color of the protein.
• Light energy induces conformational changes in the cofactor allowing for 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 Details

• 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 the 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 but is also essential for the regulation of activity of membrane proteins and downstream signaling 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.