Protein Structure and Machines

Protein Structure

• Primary structure is the amino acid sequence of a protein.
• Secondary structure arises from the folding and twisting of the amino acid chain, forming alpha-helices and beta-sheets.
• Tertiary structure is determined by hydrophilic and hydrophobic interactions between R groups of amino acid chains.
• Quaternary structure occurs in proteins with more than one amino acid chain.
• Chaperones aid in protein folding.
• Dimerization or oligomerization gives rise to quaternary structure.

Protein Machines and Modifications

• Macromolecular complexes, or protein machines, synthesize polypeptides from mRNA.
• An example of a protein machine is a ribosome, comprising 83 proteins and 4 rRNAs.
• Multi-subunit complexes offer greater flexibility.
• Post-translational modifications (PTMs) are covalent changes to protein structure.
• PTMs can alter a protein's activity, target it for degradation, change its location, or modify its structure.
• Phosphorylation is the addition of a phosphate group by kinases.
• Ubiquitination involves adding ubiquitin chains to a protein, marking it for degradation.
• Allosteric regulation is a change in protein structure or function due to non-covalent binding by a ligand, such as calcium or another protein.
• Ca2+ changes calmodulin (CaM) tertiary structure, enabling it to bind to a target protein.
• Guanosine-triphosphate (GTP) binding alters protein structure, increasing enzymatic activity.
• Guanine nucleotide exchange factor (GEF) replaces GDP with GTP.
• GTPase Activating Protein (GAP) increases GTPase activity, turning the system off.

Nucleus: Structure and Function

• The nucleus controls gene expression (transcription), which determines cell and organism characteristics.
• The nuclear envelope consists of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM), with the ONM continuous with the rough endoplasmic reticulum.
• The nuclear lamina, a protein filament network, supports the nucleus's shape.
• Nucleoli are the sites of ribosome biogenesis, forming around DNA regions encoding rRNA.
• Nuclear bodies are dynamic sub-compartments involved in transcriptional and RNA processing activity.
• Chromatin, made of DNA, RNA, and histones, determines gene expression.

Chromatin and Transcriptional Machinery

• Histone tails can undergo post-translational modifications (PTMs).
• Unacetylated chromatin is condensed and transcriptionally inactive, forming heterochromatin.
• Acetylated chromatin is less condensed and transcriptionally active, forming euchromatin.
• Proteins modifying histones control chromatin structure and DNA access for replication, transcription, and repair.
• Transcriptional activators bind to DNA, recruiting chromatin remodeling complexes to open up chromatin structure.
• A protein bridge (mediator) is recruited to help transcription factors bind to a promoter sequence.
• The mediator complex facilitates the assembly of the pre-initiation complex, including loading RNA polymerase II on DNA.
• Transcription pauses after initiation due to an elongation factor complex (NELF/DSIF).
• The elongation pause is relieved by phosphorylation and remodeling of elongation factors by a cdk/cyclin pair (P-TEFb).

Ribosome Biogenesis and Nuclear Pore Complex (NPC)

• Ribosomal RNA is transcribed by RNA Polymerase I as pre-rRNA, then processed into 28S, 18S, and 5.8S mature rRNA.
• 60S and 40S ribosome subunits assemble into functional 80S translation machinery in the cytoplasm.
• The Nuclear Pore Complex (NPC) is a large structure of nucleoporins spanning the nuclear envelope.
• Ions and small molecules diffuse through NPCs, while larger proteins and ribosomal subunits are selectively transported.
• NPCs have 8-fold spoke symmetry, cytoplasmic and nuclear asymmetry, and a flexible central channel containing FG-Nups to facilitate binding of transporters and cargo.

Nuclear Transport Mechanism

• Nuclear transport requires a localization signal on the cargo:
  • Import Signal: Nuclear Localization Sequence (NLS) is needed for import.
  • Export Signal: Nuclear Export Sequence (NES) is needed for export.
• Delivery/Transport Systems recognize localization signals on cargo to form a cargo complex.
• Importins are transporters for nuclear import.
• Exportins are transporters for nuclear export.
• Ran-GTP/GDP is the key GTPase switch in nuclear transport
• Ran is a small G-protein.
• Both Ran-GTP and Ran-GDP can diffuse through the nuclear pore

Nuclear Import Mechanism steps

• Ran-GTP binds importins, causing them to release cargo.
• Ran-GTP/Importin diffuse back into the cytoplasm.
• Cytoplasmic GAP activity converts Ran-GTP to Ran-GDP, decreasing affinity and releasing importins.
• Importins are recycled to transport more cargo.
• Ran-GDP diffuses back into the nucleus and is converted into Ran-GTP by nuclear GEFs.

RAN-GTP/GDP gradient

• High Ran-GDP concentration in cytoplasm (due to GAP).
• High Ran-GTP concentration in nucleus (due to GEF).

Ran-dependent nuclear export mechanism

• Ran-GTP binding of exportins promotes its association with cargo.
• Hydrolysis of Ran-GTP to Ran-GDP in the cytoplasm releases the exportin and cargo.
• Exportins and Ran-GDP move back through the NPC and reset by nuclear GEFs.

Nuclear Transport Components

• Bidirectional: Ran (small enough to diffuse through the pore), Transporters (Importin/Exportin, depending on how it binds FG-Nups).
• Unidirectional: Cargo (NLS or NES, due to the gradient of Ran-GTP and its interaction with transporters).
• Laminopathies are genetic mutations affecting lamins, nuclear membrane proteins connected to lamins, or proteins involved in lamin processing.

Endoplasmic Reticulum (ER)

• The secretory pathway starts at the rough endoplasmic reticulum (RER).
• In the RER, nascent proteins are folded, modified, and assembled within the ER lumen.
• The RER has a sheet-like structure called cisternae.
• The smooth endoplasmic reticulum (SER) has a highly branched, tubular morphology with 3-way branching
• Reticulons are ER membrane proteins that curve the membrane.
• Dimerization of Atlastin-GTP connects opposite membranes, causing 3-way branching.
• Cotranslational translocation is the simultaneous transport of a secretory protein into the ER as it is being synthesized.

Signal Recognition Particle (SRP) and Protein Insertion

• SRP stands for Signal Recognition Particle.
• SRP binds to the large ribosomal subunit and the signal sequence of the growing peptide.
• SRP binds to the SRP receptor, opening a channel for the translocation of the newly synthesized peptide.
• Signal peptidase in the ER cleaves the signal sequence off the polypeptide.
• Insertion of Type 1 membrane proteins requires a "stop-transfer anchor" (STA) signal.
• Type 1 membrane proteins have a cleavable signal sequence, with the N terminus inside the lumen and the C terminus in the cytosol.
• STA is a hydrophobic stretch of amino acids (20-25 aa) that embeds into the lipid bilayer.
• Insertion of Type 2 membrane proteins:
  • An internal ER targeting sequence is recognized by SRP and directed to the ER translocon.
  • The internal targeting signal also acts as a signal-anchor sequence (SA).
  • Positive ions are located before the signal-anchor sequence.
• Type 2 membrane proteins have a signal-anchor sequence, with the C-terminus inside the lumen and the N-Terminus in the cytosol.
• Insertion of Type 3 membrane proteins:
  • Uses a signal-anchor sequence positioned close to the N-terminus.
  • Positive ions follow the signal-anchor sequence.
• Type 3 membrane proteins have the N-terminus inside the lumen and the C-terminus in the cytosol, with the signal anchor sequence close to the N-terminus.
• Type 4 membrane proteins are G-protein coupled receptors.

ER Protein Modifications

• ER protein modifications include glycosylation, proteolysis, disulfide bond formation, and quaternary structure formation.
• Glycosylation is the transfer of a chain of sugars (glycans) from a precursor, catalyzed by glycosyltransferases.
• Glycosylation aids in protein folding and can determine protein function.
• Spastic Paraplegia is an example of an ER disease where mutations impact reticulons and atlastin.

Golgi Apparatus

• The Golgi apparatus is organized around the centrosome/Microtubule Organizing Centre (MTOC).
• The Golgi receives newly synthesized and correctly assembled secretory cargo proteins from the ER.
• The Golgi structure consists of flattened stacked disks called cisternae.
• Golgi organization includes the cis Golgi Network (CGN), medial cisternae, and trans Golgi Network (TGN).
• GRASPs (Golgi Reassembly and Stacking Proteins) are membrane-associated proteins that dimerize/oligomerize, stacking parallel cisternae together.
• Golgins are coiled-coil proteins with an extended rod-like conformation (tethering).

Microtubule Network and Golgi

• The microtubule network helps to: 1. Maintain ribbon structure. 2. Maintain Perinuclear localization.
• During mitosis, the Golgi temporarily fragments into mini-stacks and individual cisternae.
• Formation of the Golgi Ribbon involves clustering of mini-stacks at the perinuclear region, tethering by golgins, and membrane fusion by SNAREs.
• COPII vesicles move from the ER to the Golgi in anterograde transport.
• COPI vesicles move from the Golgi to the ER in retrograde transport.
• Sorting signals on the cytoplasmic domain of membrane cargo proteins are recognized by coat proteins for inclusion in budding vesicles.
• Soluble cargo proteins with luminal sorting signals require recognition by membrane cargo receptors for transport selection.

KDEL Receptor

• KDEL receptor is the sorting signal for retrieving ER-resident proteins.
• It is activated in the Golgi but deactivated in the ER.
• KDEL receptors bind KDEL sequences in the Golgi due to a slight pH difference.

Glycosylation in the Golgi

• Glycoproteins are proteins modified with sugar polymers or glycans.
• Glycosidases remove sugars from sugar chains on glycoproteins.
• Glycosyltransferases add individual sugars to sugar chains.
• The three compartments of the Golgi apparatus are cis-, medial-, and trans-.
• Cis-Golgi glycans become substrates for medial-Golgi enzymes.
• Medial-Golgi glycans become substrates for trans-Golgi enzymes.

Golgi Enzymes and Compartments

• Different sugar-modifying enzymes modify glycans in cis-, medial-, and trans- compartments.
• Distinct sugar-modifying enzymes are retained in the Golgi via COPI retrograde transport to newer cisternae as older cisternae transition.