p53 Protein and Structure

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Questions and Answers

What is a characteristic feature of the leucine zipper motif in eukaryotic transcription factors?

Contains no leucine residues

Always begins with a charged residue

Has a highly conserved DNA binding region (correct)

Contains only hydrophobic amino acids

Which amino acid position in the leucine zipper motif is always leucine?

Third position (c)

First position (a)

Fourth position (d) (correct)

Seventh position (g)

What structure does a leucine zipper typically form?

Parallel coiled-coil alpha helices (correct)

Linear polypeptide chains

Globular protein structure

Single stranded helix

In which dimerization scenario do two identical transcription factors interact?

Homodimer Signup and view all the answers

Which transcription factor cannot form a homodimer due to charge repulsion?

Fos Signup and view all the answers

What type of side chains are typically found outside the hydrophobic core in leucine zippers?

Charged and can affect dimer formation Signup and view all the answers

How often does the structure of a leucine zipper repeat in terms of residues?

Every 7 residues Signup and view all the answers

Which mammalian transcription factor is classified as a proto-oncogene?

Jun Signup and view all the answers

What does the 'p' in p53 stand for?

Protein Signup and view all the answers

What is a key role of the p53 protein in cell growth?

Controls a critical step in the cell cycle Signup and view all the answers

Which domain is primarily responsible for the dimer-dimer interactions in the p53 tetramer formation?

Oligomerization domain Signup and view all the answers

How does p53 contribute to the cell cycle in the presence of damaged DNA?

It initiates programmed cell death (apoptosis) Signup and view all the answers

Which of the following is true regarding the structure of p53?

Involves beta strands and alpha helices arranged in a specific motif Signup and view all the answers

What type of motifs are associated with specific transcription factors related to DNA complexes?

Leucine zipper and helix-loop-helix Signup and view all the answers

What happens to tumor-derived p53 mutants regarding DNA binding?

They are defective in sequence-specific DNA binding Signup and view all the answers

What is the role of p21 in the cell cycle in relation to p53?

Halts the cell cycle before division Signup and view all the answers

What facilitates the formation of heterodimers between Fos and Jun?

Complementary positive charges in specific positions Signup and view all the answers

How many distinct DNA-binding specificities can three types of monomers produce?

6 Signup and view all the answers

How does the binding affinity of the Fos-Jun heterodimer compare to the Jun homodimer?

It binds with 10 times higher affinity Signup and view all the answers

What is the length of the monomer GCN4 in amino acids?

281 aa Signup and view all the answers

In GCN4, what is the main function of the basic region?

To bind to specific promoter regions Signup and view all the answers

What is the length of the basic region in GCN4?

20 aa Signup and view all the answers

What type of sequences do GCN4 monomers bind to?

Pseudopalindromic nucleotide sequences Signup and view all the answers

What characterizes the leucine zipper region in GCN4?

It packs into a coiled coil Signup and view all the answers

What does G protein bind to in order to become active?

GTP Signup and view all the answers

What happens to G protein when GTP is replaced by GDP?

It becomes inactive. Signup and view all the answers

Which subunit of the G protein possesses GTPase activity?

Alpha subunit Signup and view all the answers

What is the primary role of G proteins in cellular signaling?

To amplify signals. Signup and view all the answers

What occurs when RGS binds to G proteins?

It switches off gene activation. Signup and view all the answers

Which configuration allows Gα to remain monomeric?

When bound to GTP. Signup and view all the answers

How many different genes code for G protein coupled receptors?

1000 Signup and view all the answers

What happens to Gα, Gβ, and Gγ species upon activation of the G protein?

Gα dissociates from GβGγ. Signup and view all the answers

What is formed during the cleavage of the peptide bond in the first step of the reaction?

Acyl-enzyme intermediate Signup and view all the answers

What role does the negatively charged tetrahedral transition state intermediate play in the reaction?

It facilitates the hydrolysis of the acyl-enzyme intermediate. Signup and view all the answers

Which amino acids compose the catalytic triad in the serine protease mechanism?

Asp-His-Ser Signup and view all the answers

What primary function does the oxyanion hole serve in serine proteases?

It stabilizes the transition state during catalysis. Signup and view all the answers

How does Trypsin achieve specificity for its substrates?

By favoring positively charged residues. Signup and view all the answers

In the context of mutational effects, what impact does changing Gly 216 to Ala 216 have on Trypsin?

It displaces a water molecule that binds to the substrate. Signup and view all the answers

What distinguishes Chymotrypsin from other serine proteases in terms of substrate specificity?

It cleaves at aromatic residues. Signup and view all the answers

What is the structural significance of the two domains in Chymotrypsin?

They hold the polypeptides together by disulphide bridges. Signup and view all the answers

Study Notes

p53 Protein

p53 protein plays a critical role in human cell growth
Mutations in p53 protein are linked to tumor formation
p53 maintains genome integrity during cell division
p53 controls a crucial step in the cell cycle
p53 interacts with cyclins and cyclin-dependent kinases (CDKs)
p53 promotes the expression of p21, which inhibits CDKs
p21 halts the cell cycle before cell division
p53 allows the cell to repair damaged DNA or initiate apoptosis
Wild-type p53 binds to specific DNA sequences
Tumor-derived p53 mutants are defective in DNA binding, preventing gene activation.

p53 Structure

p53 has three domains: N-terminal activation domain, DNA-binding domain, and C-terminal oligomerization domain.
The oligomerization domain forms tetramers.
The oligomerization domain is comprised of a 32-amino acid peptide.
The structure of each oligomerization domain unit is a beta-strand-turn-alpha-helix motif.
Beta strands form an antiparallel two-stranded beta-sheet.
Alpha helices are arranged in an antiparallel fashion with eight backbone hydrogen bonds.
Tetramers form through hydrophobic interactions between alpha helices.
Beta strands are not involved in dimer-dimer interactions.
The arrangement of four alpha helices packed against each other is unusual.
Mutations in the oligomerization domain, specifically in the L2 and L3 loops, can distort the structure and affect DNA binding.

Leucine Zipper

Leucine zippers are dimerization domains found in bZIP (basic-region leucine zipper) eukaryotic transcription factors.
bZIP domains are 60-80 amino acids long, containing a conserved DNA-binding basic region and a leucine zipper dimerization region.
Leucine zippers were first recognized in the yeast transcription factor GCN4.
Other examples include the mammalian transcription factor C/EBP and proto-oncogenic transcription factors Fos, Jun, and Myc.
When plotted in a helical wheel, leucine residues in linear amino acid sequences form a distinct pattern.
The leucine zipper region consists of repeating units of seven amino acids, with the fourth residue always being a leucine.
The first residue in each unit is usually hydrophobic.
Dimerization forms two parallel coiled-coil alpha helices with a helical repeat of 3.5 residues per turn.
The hydrophobic core region is formed by interactions between leucine residues at positions "a" and "d" in each unit.
Charged residues at "e" and "g" positions influence dimer formation, either promoting or preventing it based on charge interactions.

Leucine Zipper Dimerization

Leucine zippers can form homodimers (same transcription factors) or heterodimers (different transcription factors).
Fos/Jun heterodimer is found in AP1 (Active gene regulating protein 1), responsible for cell proliferation.
Jun can form both homodimers and heterodimers.
Fos cannot form homodimers due to strong charge repulsion from glutamic acid residues at "e" and "g" positions, lacking compensating positive charges.
Fos can form heterodimers with Jun due to complementary positive charges in the "e" and "g" positions of Jun.
Heterodimer formation expands the repertoire of DNA-binding specificities.
Two types of monomers can generate three distinct DNA-binding specificities.
Three types of monomers can form six distinct DNA-binding specificities.

Leucine Zipper and DNA Binding

The Fos-Jun heterodimer binds to DNA with the same target specificity as the Jun homodimer but with 10 times higher affinity to the AP1 binding site.
The ability of leucine zipper proteins to form heterodimers expands their DNA-binding specificities.

GCN4 bZIP Transcription Factor

GCN4 is a yeast transcription factor containing a basic region-leucine zipper (bZIP) motif.
GCN4 monomer is 281 amino acids long.
GCN4 binds to promoter regions of genes involved in amino acid biosynthesis, especially during amino acid starvation.
The dimerization and DNA binding domains are located in separate regions: the basic region and the C-terminal leucine zipper region (totaling 55 amino acids).
The DNA recognition region of GCN4 is similar to the Fos/Jun heterodimer of AP1.

GCN4 Structure and DNA Binding

The basic region of GCN4 is approximately 20 amino acids long and contains eight charged residues, mainly arginine, involved in DNA binding.
The basic region is disordered in solution without DNA.
The GCN4 bZIP region complexed with a 20 base pair DNA fragment containing a pseudo-palindromic sequence has been studied.
Each GCN4 monomer forms a curved, continuous alpha helix.
The leucine zipper region of the monomers packs into a coiled coil.
The GCN4 structure is too large for NMR analysis.

G Protein-Coupled Receptors (GPCRs)

GPCRs are transmembrane proteins with six helices.
GPCRs amplify signals transmitted from the extracellular domain to the intracellular domain.
GPCRs use G proteins as signal amplifiers.
G proteins bind to guanine nucleotides, hence the name.
G proteins function as molecular switches:
- Active state: G protein + GTP
- Inactive state: G protein + GDP
G proteins have slow GTPase activity.
RGS (Regulators of GTP Hydrolysis) turn off gene activation by binding to G proteins.
Active G proteins (GTP-bound) can activate multiple downstream effectors, amplifying the signal.
RGS binding switches off the signal.

G Protein Subunits

G proteins are heterotrimers, consisting of alpha, beta, and gamma subunits.
There are 1000 different genes coding for GPCRs, leading to a variety of G proteins with different alpha, beta, and gamma subunits.
The alpha subunit contains GTPase activity.
The inactive state of a G protein is Gα-GDP-GβGγ.
External signals activate GPCRs, triggering conformational changes in the cytosolic domain.
Activated GPCRs trigger the release and dissociation of Gα-GTP.
All three G protein species (Gα, GβGγ, GαGβGγ) are attached to the cell membrane via lipid modifications of alpha and gamma subunits.
Gα-GDP forms a complex with GβGγ, while Gα-GTP is monomeric.

Chymotrypsin Superfamily

The chymotrypsin superfamily includes chymotrypsin, trypsin, thrombin, and elastin.
Bacterial serine proteases are also part of this family.
Despite different sequences and structures, the chymotrypsin superfamily members share a common active site architecture, indicating convergent evolution.
Common features include:
- Catalytic triad (Asp-His-Ser)
- Oxyanion hole
- Substrate binding site
This shared active site architecture reflects a structural solution for achieving a specific catalytic mechanism.
Subtilisin, a bacterial serine protease, is structurally distinct from mammalian serine proteases but shares the same active site architecture.

Chymotrypsin Structure

Chymotrypsin undergoes a conversion process from chymotrypsinogen (254 amino acids) to chymotrypsin.
During conversion, two peptide segments (residues 14-15 and 147-148) are excised.
The resulting chymotrypsin consists of three polypeptides linked by disulfide bridges.
Chymotrypsin has two domains with similar structures, each containing about 120 amino acids.
Each domain is an antiparallel beta barrel with six beta strands and a Greek key motif (1-4) followed by a hairpin motif.

Chymotrypsin Active Site

The active site of chymotrypsin is located in a crevice between domain 1 and domain 2.
Key residues in the active site include:
- Histidine 57 (domain 1)
- Aspartate 102 (domain 1)
- Serine 195 (domain 2)

Substrate Specificity Pocket

Different proteases cleave polypeptide chains at specific residues based on their substrate specificity pockets.
Chymotrypsin has a wide pocket that accommodates large aromatic side chains.
Trypsin has a positively charged specificity pocket due to Asp, Lys, and Arg residues, allowing it to bind to positively charged Lys and Arg residues in substrates.
Elastase has a small hydrophobic pocket that prefers small hydrophobic residues.

Mutational Effects on Trypsin

Replacing Gly 216 with Ala 216 in the specificity pocket of trypsin displaces a water molecule that normally binds to the NH3+ group of a substrate's lysine side chain.
This displacement alters the specificity of the mutant enzyme.

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Explore the crucial role of p53 protein in cell growth, tumor suppression, and DNA repair mechanisms. This quiz covers the structure of p53, its interaction with various cellular components, and the significance of its mutations in cancer development.