Molecular Basis for the Interaction Between AP4 beta 4 and its Accessory Protein, Tepsin

The adaptor protein 4 (AP4) complex (E/4/4/sigma 4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal 4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the 4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on 4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4-coated vesicles in cell biology, and especially in neuronal function

Molecular Basis for the Interaction Between AP4 β4 and its Accessory Protein, Tepsin.

The adaptor protein 4 (AP4) complex (ϵ/β4/μ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 β4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal β4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the β4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on β4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4-coated vesicles in cell biology, and especially in neuronal function

Structural Perturbations Induced by the α-Anomer of the Aflatoxin B₁ Formamidopyrimidine Adduct in Duplex and Single-Strand DNA

The guanine N7 adduct of aflatoxin B₁ <i>exo</i>-8,9-epoxide hydrolyzes to form the formamidopyrimidine (AFB-FAPY) adduct, which interconverts between α and β anomers. The β anomer is highly mutagenic in <i>Escherichia coli</i>, producing G → T transversions; it thermally stabilizes the DNA duplex. The AFB-α-FAPY adduct blocks replication; it destabilizes the DNA duplex. Herein, the structure of the AFB-α-FAPY adduct has been elucidated in 5′-d(C¹T²A³T⁴<u>X</u>⁵A⁶T⁷T⁸C⁹A¹⁰)-3′·5′-d(T¹¹G¹²A¹³A¹⁴T¹⁵C¹⁶A¹⁷T¹⁸A¹⁹G²⁰)-3′ (X = AFB-α-FAPY) using molecular dynamics calculations restrained by NMR-derived distances and torsion angles. The AFB moiety intercalates on the 5′ face of the pyrimidine moiety at the damaged nucleotide between base pairs T⁴·A¹⁷ and X⁵·C¹⁶, placing the FAPY C5−<i>N</i>⁵ bond in the <i>R</i>_<i>a</i> axial conformation. Large perturbations of the ε and ζ backbone torsion angles are observed, and the base stacking register of the duplex is perturbed. The deoxyribose orientation shifts to become parallel to the FAPY base and displaced toward the minor groove. Intrastrand stacking between the AFB moiety and the 5′ neighbor thymine remains, but strong interstrand stacking is not observed. A hydrogen bond between the formyl group and the exocyclic amine of the 3′-neighbor adenine stabilizes the <i>E</i> conformation of the formamide moiety. NMR studies reveal a similar 5′-intercalation of the AFB moiety for the AFB-α-FAPY adduct in the tetramer 5′-d(C¹T²<u>X</u>³A⁴)-3′, involving the <i>R</i>_<i>a</i> axial conformation of the FAPY C5−<i>N</i>⁵ bond and the <i>E</i> conformation of the formamide moiety. Since in duplex DNA the AFB moiety of the AFB-β-FAPY adduct also intercalates on the 5′ side of the pyrimidine moiety at the damaged nucleotide, we conclude that favorable 5′-stacking leads to the <i>R</i>_<i>a</i> conformational preference about the C5−<i>N</i>⁵ bond; the same conformational preference about this bond is also observed at the nucleoside and base levels. The structural distortions and the less favorable stacking interactions induced by the AFB-α-FAPY adduct explain its lower stability as compared to the AFB-β-FAPY adduct in duplex DNA. In this DNA sequence, hydrogen bonding between the formyl oxygen and the exocyclic amine of the 3′-neighboring adenine stabilizing the <i>E</i> configuration of the formamide moiety is also observed for the AFB-β-FAPY adduct, and suggests that the identity of the 3′-neighbor nucleotide modulates the stability and biological processing of AFB adducts

Accurate protein-peptide titration experiments by nuclear magnetic resonance using low-volume samples

AGAP : équipe IDInternational audienceNMR spectroscopy allows measurements of very accurate values of equilibrium dissociation constants using chemical shift perturbation methods, provided that the concentrations of the binding partners are known with high precision and accuracy. The accuracy and precision of these experiments are improved if performed using individual capillary tubes, a method enabling full automation of the measurement. We provide here a protocol to set up and perform these experiments as well as a robust method to measure peptide concentrations using tryptophan as an internal standard