Patients with
Alport syndrome (AS) exhibit blood and elevated
protein levels in their urine, inflamed kidneys, and many other abnormalities. AS is attributed to mutations in
type IV collagen genes, particularly
glycine missense mutations in the collagenous domain of COL4A5 that disrupt common structural motifs in
collagen from the repeat (Gly-Xaa-Yaa)n amino acid sequence. To characterize and elucidate the molecular mechanisms underlying how AS-related mutations perturb the structure and function of
type IV collagen, experimental studies and molecular simulations were integrated to investigate the structure, stability,
protease sensitivity, and
integrin binding affinity of
collagen-like
proteins containing amino acid sequences from the α5(IV) chain and AS-related Gly missense mutations. We show adverse effects where (i) three AS-related Gly missense mutations significantly reduced the structural stability of the
collagen in terms of decreased melting temperatures and calorimetric enthalpies, in conjunction with a collective drop in the external work needed to unfold the
peptides containing mutation sequences; (ii) due to local unwinding around the sites of mutations, these triple helical
peptides were also degraded more rapidly by
trypsin and
chymotrypsin, as these
enzymes could access the collagenous triple helix more easily and increase the number of contacts; (iii) the mutations further abolished the ability of the recombinant
collagens to bind to
integrins and greatly reduced the binding affinities between
collagen and
integrins, thus preventing cells from adhering to these mutants. Our unified experimental and computational approach provided underlying insights needed to guide potential
therapies for AS that ameliorate the adverse effects from AS disease onset and progression.