nucleic lec 6

Protein Function

• Proteins have a vast array of functions in nature, arising from a combination of factors.
• These factors include recognizing and binding other molecules (including proteins, nucleic acids, and small molecules).
• They also catalyze chemical reactions, acting as enzymes.
• Proteins can serve as substrates for modifications.
• Combined with intrinsic structure and dynamics, these functions can result in complex roles.
• Hemoglobin, for example, binds heme, reacts with CO₂, binds bisphosphoglycerate, and binds O₂, enabling oxygen transport throughout the body.

Binding

• Cells contain numerous molecules like ions, proteins, nucleic acids, lipids, and metabolites.
• These molecules constantly diffuse and collide within the cell (in bulk solution or within a membrane).
• Individual proteins recognize specific molecules from this complex mixture.
• Recognition implies a stable protein complex facilitating a biologically relevant effect.
• These complexes form the basis for signaling events, catalysis, and protein localization.

Binding Energetics

• Binding requires a lower energy state for the complex compared to isolated molecules.
• Driving forces of binding are hydrogen bonds, van der Waals interactions, and hydrophobic interactions.
• The energy required to displace water and loss of conformational heterogeneity contribute to a balanced energy equation.
• Binding interaction generally maximizes the contact surface area.

Stable vs. Transient Interactions

• Stable interactions (in a long period) exhibit micromolar or better affinity (even picomolar in certain cases).
• Other processes require weak and fleeting interactions, typically within the micromolar to millimolar range, and a large fraction of the protein may remain unbound at any given time.

Small Molecule Binding

• Cells contain diverse small (non-polymer) molecules like metabolites, building blocks, enzyme cofactors, and ions.
• The binding site of a protein designed to recognize a specific small molecule is pre-organized to specifically bind that ligand.
• This binding site is generally complementary to the ligand's properties.

Streptavidin

• Streptavidin is a 159-amino acid protein from Streptomyces avidinii.
• It acts as a biotin-scavenging protein (a crucial cofactor in carboxylation reactions like acetyl-CoA carboxylase in fatty acid synthesis).
• Streptavidin exhibits exceptionally high affinity for biotin (~10^-14 mol/L).
• It's widely used in biotechnology for reliably capturing biotin-tagged molecules, even at low concentrations.

Streptavidin Structure

• Streptavidin consists of eight β-strands forming a beta sheet (up-down topology), with the first and last strands forming a beta barrel.
• The functional tetramer originates from four streptavidin protomers in D₂ symmetry.

Streptavidin Binding Biotin

• Biotin binds each protomer in the center of the beta barrel.
• The apo structure's short helix rearranges into a loop to encapsulate biotin during binding.
• During binding, typical structural transitions are observed.

Biotin Binding Details

• Biotin's binding site is primarily non-polar.
• Polar groups within the site are buried and stabilized by hydrogen bonds.
• The hydrophobic effect contributes towards biotin binding.

Biotin Shape Complementarity

• The streptavidin binding pocket precisely matches biotin's shape.
• Nonpolar atoms are in close van der Waals contact.
• Polar atoms are closer, forming shorter hydrogen bonds.

Protein-Protein Binding

• Proteins can form both permanent and short-lived complexes.
• Complexes often localize proteins within cells, modulate activity, or serve substrates/allosteric modulators for each other.
• Protein-protein interactions typically involve extensive buried surface areas (minimum 700 Ų) for strong binding.
• The interactions are usually similar to oligomeric interfaces, but potentially less hydrophobic if the complexes are temporary.

Ran and RanGAP Complex

• Ran and RanGAP form a short-lived signaling complex.
• Ran (purple) and RanGAP (grey) promote GDP-GTP exchange, leading to Ran's active site.
• Individual proteins fold independently but transiently associate..
• Proteins do not extensively reorient, the Ran active site only slightly restructures to promote GDP exchange.
• Extended protein surfaces are buried between the proteins.

Ankyrin Complex in Erythrocytes

• Ankyrin is a large helical repeat protein (in red).
• It links the cytoskeleton to the cell membrane via proteins like spectrin.
• In erythrocytes, it binds the regulatory domains of proteins like bicarbonate/chloride antiporter, Rh protein, and aquaporin.
• The complex specifically anchors the cytoskeleton to the membrane.

Protein-Peptide Binding

• Peptide binding differs from protein binding because peptides lack intrinsic structure.
• This allows peptides to conform to the target protein surface.
• Maximizing interactions comes at a cost, losing conformational entropy during binding.

Nucleic Acid Binding

• Nucleic acid binding proteins mostly use multiple basic residues (arginine) to interact with the polyphosphate backbone.
• Hydrogen bonds to exposed polar groups add specificity.
• For RNA binding, stacking on exposed bases can contribute to stability.
• Unstructured positive-charge-rich regions becoming ordered upon NA binding is common.

Enzyme Catalysis

• Many proteins act as catalysts accelerating chemical reactions.
• Most enzyme catalytic mechanisms are either acid-base chemistry (transferring protons), or redox chemistry (transfer of electrons or hydride ions).
• Enzymes utilize multiple groups for acid-base chemistry, or external factors for redox chemistry.

Enzyme Active Sites

• Enzymes bind substrates in conformations optimized for the reaction.
• Catalytic residues, like polarizing bonds and attacking reaction centers, are nucleophilically or electrophilically, and/or remove or add protons or electrons.

Catalysis and Amino Acids

• Catalysis heavily relies on a few charged or titratable amino acids.
• Histidine is a commonly used catalytic residue even though not especially frequent.
• Catalytic residues are typically charged or possess a pKa near neutrality.
• Polar side chains are more frequently involved than nonpolar aliphatic side chains (like glycine, alanine, valine, isoleucine, leucine, proline).

Protein Cofactors

• Enzymes and other proteins use diverse cofactors (e.g., pyridoxal phosphate, biotin, SAM).
• Cofactors help perform chemically challenging tasks that amino acid side chains can’t.
• Associated cofactors (tightly bound or covalently linked) remain permanently.
• Cofactors can enable proteins to capture energy (light, e.g., chlorophyll) or utilise challenging groups (e.g., heme, biotin with CO2, cobalamin with CH3, FAD with H-, pyridoxal phosphate with NH3).
• Cofactors can transport activated chemical groups between enzymes, like hydride (NADPH) and methyl (SAM).

D-Lactate Dehydrogenase

• D-lactate dehydrogenase reduces pyruvate to D-lactic acid (redox reaction).
• In bacteria, it is crucial in serine metabolism, as is a human-equivalent protein..
• The active site is located in the cleft between two Rossmann domains.

DLDH Catalytic Groups

• Proteins lack good hydride donors, so NADH provides hydride.
• Proton transfer happens from histidine to pyruvyl groups; glutamate participates in histidine pKa shift.
• Arginine is vital for polarising and activating the keto group.
• These elements are essential and conserved amongst DLDH family in similar reactions.

D-LDH Domain Closure

• D-LDH's small domain closure isolates substrates to ensure substrates cannot bind.

D-Lactate Dehydrogenase Binding

• D-LDH binds a small substrate (pyruvate) enabling specificity by making favourable contacts with every atom.
• Pyruvate stacks on the nicotinamide ring of NAD.
• Catalytic residues contribute to binding.
• Catalytic arginine and histidine form hydrogen bonds.

D-LDH Carboxylate Binding

• Substrate carboxylate group is bound via hydrogen bonds from backbone and ligands.
• Two backbone amide nitrogens bind the substrate carboxylate.
• Tyrosine contributes to binding with a third hydrogen bond.

D-LDH Substrate Specificity

• The methyl group within D-LDH is located in a hydrophobic pocket with Phe and Tyr.
• Van der Waals interactions with other metabolites resemble pyruvate (but differe).
• Phe and Tyr residues crucially maintain specificity.

Metal Ions

• Many proteins bind specific metal ions for catalysis (e.g., metalloproteases), structure (e.g., zinc finger proteins), or regulation (e.g., calmodulin).
• Metal ions are coordinated by conserved amino acids (Cys, His, Asp, or Glu; H₂O or S).
• Transition metal ion-protein bonds are almost covalent.
• Transition metals are fundamental participants in redox reactions.
• Iron-sulfur clusters of proteins support electron transfer over longer distances.

Src Kinase ATP Binding

• Src kinase binds ATP in a pre-organized pocket.
• Hydrophobic residues in the pocket bind to adenine base + ribose.
• Hydrogen bonds connect to backbone and help neutralize the PO₄ group .
• A magnesium ion assists phosphate coordination.
• Water molecules are essential for interactions with ATP.

Calmodulin

• Calmodulin is a calcium-modulated protein responding to elevated intracellular calcium levels.
• It binds to diverse targets (over 300).
• Structures of two distinct EF-hand 4 helix bundles joined by a linker.

Calmodulin EF Hands

• EF hands are helix-turn-helix motifs.
• The turn regions (white) are relatively long and include important amino acids like Asp and Glu, at the end of the second helix.
• These residues are essential for calcium binding.
• Similar motifs are found in many other proteins.

Calcium Binding

• One calcium ion binds to each loop region in helix-loop-helix.
• The loop flexibility orients residues towards calcium.
• Calcium prefers oxygen ligands (especially carboxylates).
• Calcium is coordinated between 2x Asp, Glu, Asn plus a backbone carbonyl oxygen.

Calcium Binding Structural Change

• To bind calcium, helices repack, changing arrangement from almost parallel to at right angles.

Ca-Calmodulin Complex Hydrophobic Patch

• Rearrangement opens a hydrophobic core within the EF hand.
• This exposes a hydrophobic surface.
• The favoured energy from calcium binding compensates for exposing hydrophobic residues to water.

Ca-Calmodulin Protein Binding

• Ca-Calmodulin binds diverse targets (with exposed nonpolar regions.
• EF hands adjust relative position and shape of the cavity.
• Methionine positions increase flexibility to optimise packing.
• Calmodulin regulates over 300 different proteins, including a calmodulin-dependent kinase.
• Forming this complex activates the kinase.

Covalent Modifications

• Over 200 covalent modifications are common in eukaryotic proteins, including glycosylation for signal transduction, lipidation for membrane localization, and ubiquitination for degradation.

Protein Glycosylation

• Extracellular domains in eukaryotes often have heavily-modified oligosaccharides (up to 20% protein mass).
• Glycosylation affects cellular trafficking, and are targets for immune interactions.
• Saccharides are added to specific sites (N-linked to Asn; O-linked to Ser/Thr).

Regulatory Modifications

• Eukaryotic proteins undergo regulatory modifications by small molecular groups.
• This alters activity and interaction with other proteins. –e.g. phosphorylating Tyr, Ser, Thr residues (kinase action); acetylating Lys (on histones).
• Modifications may employ whole protein units (e.g. ubiquitin, or SUMO).
• Lipid addition can temporarily or permanently target proteins to the membrane.

Function and Structure Constraints

• Protein function and structure are related (constraining their sequences).

Protein Mutations

• Protein coding sequences are susceptible to mutations.
• Mutations from mutagens or DNA replication errors accumulate slowly, even within a species.
• Amino acid sequence differences can be present between proteins from different species.
• Mutations are often neutral, however, they can impact protein function in the event more apparent differences are found. Evolutionary changes are not randomly distributed.

Active Site Residues

• Active site residues are crucial for function and highly conserved.
• An enzyme's active site is complementary to the ligand, facilitates catalysis.
• Important residues are conserved, and if mutated could reduce efficiency.
• Even minor alterations in the specific amino acid sequence can affect substrate specificity.

Buried Residues

• Buried residues affect protein stabilization and key element positioning.
• Commonly conserved.
• Tolerates substitutions of similar amino acids (e.g. Val to Ile).

Surface Residues

• Surface residues (not involved in catalysis or binding) are often more variable.
• Surface residues are predominantly involved in protein-protein interactions.
• Substitutions are generally tolerated and specific to protein solvent interactions.

Different Sequences for Protein Function

• Many residues that don't directly affect function can drift over generations resulting in slight differences in amino acid sequence even within species.
• For instance, hen and turkey lysozymes differ by just 5 amino acids, but the main functions are similar. This is an example of differing sequence not substantially impacting the final outcome.

Protein Divergence

• Proteins can maintain or acquire new functions by slowing accumulating changes over evolutionary time.
• Proteins can vary in function despite sequence similarity.
• Switching function is more probable when proteins with existing functions are interacting with only slightly differing substrates.

Substrate Specificity in D-LDH Homologs

• D-2-hydroxyisocaproate DH similar to D-LDH, but differ in the substrate it works on (resulting in different precursors).
• Changes in D-LDH’s methyl-binding pocket allow accommodation of larger substrates rather than pyruvate (by changes in amino acid structure).
• Other family members use varying groups to bind different substrates, thereby, resulting in different methyl-binding pockets.

Residues Differentiate Homologs

• Residues lining the methyl pocket are significantly different, distinguishing D-LDH orthologs from related enzymes with dissimilar substrates.

β-Carbonic Anhydrases

• Carbonic anhydrases use a zinc-activated water ion to hydrolyse CO₂, forming carbonic acids crucial in various biochemical processes.
• The catalytic zinc site comprises a conserved Cys-X-X-Cys motif with an Asp/Arg segment.
• A hydrophobic pocket helps to bind CO₂.

β-Carbonic Anhydrase Divergence

• Bacterial carbonic anhydrases (epsilon class) exhibit high sequence divergence compared to their pea counterparts (yet maintaining similar structure and active sites).
• The active site has a conserved structure although only 5 critical active site residues are highly similar across similar types of enzymes.

Human A/B/O Glycosyltransferases

• Human blood groups are determined by the presence of A/B/O antigens.
• The differences are due to different glycosyltransferases in the saccharide group (GlcNAc, Glc, or no saccharide) attached to a ceramide pentasaccharide.
• Different inactive alleles can give rise to different O-antigen blood groups

GTA vs GTB Glycosyltransferases

• These enzymes have slightly different, but highly similar, structures.
• GTA transfers N-acetyl glucosamine to the antigen, while GTB transfers glucose to the same antigen.
• Differences centre on adding the amide to C₂ in the different substrate types.

GTA vs GTB Active Site

• GTA and GTB enzymes (A and B glycolsyltransferases) differ in 4 amino acids.
• Leu to Met exchange in amino acid sequence directly impacts the space available to bind amide group.
• These minor differences result in important biochemical functional differences.

Protein Divergence and Function

• Sequence divergence in protein functional areas is associated with new function formation.
• Key changes in local areas (like DLDH) define differences in substrate recognition.
• Minor substitutions, such as in GTA and GTB, can lead to changes in function.
• Proteins can maintain their function despite notable sequence differences (e.g., in differing types of carbonic anhydrases).

Protein Function Variation with Sequence Similarity

• Proteins with the potential to evolve new functions easily show structurally or chemically similar partners.
• Minor substrate differences in an existing binding site enable a protein to function with slightly different substrates.
• Highly different substrates require many changes in the enzyme for a new function to evolve; for this reason, it is less likely to occur.

Bacterial Enzymes and Diversity

• Eukaryotic organisms exhibit similar core metabolisms and enzymatic properties.
• Bacteria display enormously diverse metabolisms, with a broader range of enzyme functions to adapt to various molecular environments.
• Repeated evolutionary mechanisms lead to evolution of new functions in bacterial enzyme families
• Function identification in bacterial enzymes can be quite challenging given their vast diversity.