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Numerous mutations in the Plasmodium falciparum Kelch13 (K13) protein confer resistance to artemisinin derivatives, the current front-line antimalarial drugs. K13 is an essential protein that contains BTB and Kelch-repeat propeller (KREP) domains usually found in E3 ubiquitin ligase complexes that target substrate protein(s) for ubiquitin-dependent degradation. K13 is thought to bind substrate proteins, but its functional/interaction sites and the structural alterations associated with artemisinin resistance mutations remain unknown. Here, we screened for the most evolutionarily conserved sites in the protein structure of K13 as indicators of structural and/or functional constraints. We inferred structure-dependent substitution rates at each amino acid site of the highly conserved K13 protein during the evolution of Apicomplexa parasites. We found two solvent-exposed patches of extraordinarily conserved sites likely involved in protein-protein interactions, one in BTB and the other one in KREP. The conserved patch in K13 KREP overlaps with a shallow pocket that displays a differential electrostatic surface potential, relative to neighboring sites, and that is rich in serine and arginine residues. Comparative structural and evolutionary analyses revealed that these properties were also found in the functionally-validated shallow pocket of other KREPs including that of the cancer-related KEAP1 protein. Finally, molecular dynamics simulations carried out on PfK13 R539T and C580Y artemisinin resistance mutant structures revealed some local structural destabilization of KREP but not in its shallow pocket. These findings open new avenues of research on one of the most enigmatic malaria proteins with the utmost clinical importance.

Although it is expected that PfK13 may act similarly to other BTB-Kelch proteins involved in ubiquitination activities17, the putative binding regions and functionally important sites of PfK13 and the structural alteration(s) associated with PfK13 ART-R mutations remain uncharacterized (Fig. 1b). Because of its high conservation9 and essentiality across Apicomplexa species11,12,13,32, the function of K13 likely remained unchanged during Apicomplexa evolution. As a consequence, we would expect that amino acid replacements at the functional sites of K13 would be strongly detrimental to protein function, and would be purged by purifying selection. To find the most conserved amino acid sites of K13, we inferred substitution rates for each amino acid site of the WT K13 sequence from 43 Apicomplexa species, taking into account both the spatial correlation of site-specific substitution rates in the protein tertiary structure and the species phylogeny33. We identified in BTB and KREP two solvent-exposed patches of extraordinarily conserved sites, likely involved in protein-protein interactions. As controls, we found similarly evolutionarily conserved and located patches in several BTB- and KREP-containing proteins found in mammals, including KEAP1. Finally, we tested through molecular dynamics simulations whether the R539T and C580Y ART-R mutations in PfK13 altered some structural properties of the KREP domain and its putative binding sites.

We then performed a more extensive study of the BTB and KREP domains of K13 because of their likely role in mediating K13 functions and the availability of their tertiary structures. To detect patches of slowly evolving amino acid sites in the BTB-KREP structure, we focused on the site-specific substitution rates λ at the amino acid level mentioned above. λ, estimated using FuncPatch, has been shown to provide a more reliable estimation of the conservation level at amino acid sites compared to standard substitution estimates, especially in the case of highly conserved proteins33,36.

Combining evolutionary and tertiary structure information provides a powerful and efficient way to gain insight into the functionality of protein sites45. People usually search for amino acid sites that have evolved more rapidly than expected under a neutral model and interpret them as a signature of adaptive evolution corresponding to a gain of new function(s)46,47. Here, we focused on the most slowly evolving, patches of amino acid sites in 3D structure to identify sub-regions of K13 that are likely to play a function conserved during the long time-period of Apicomplexa evolution. Detecting highly conserved sites in extremely conserved genes, such as pfk13, through population genetics would require a very large sampling of gene sequences48, which has not yet been reached for pfk139.

In conclusion, by comparative structural and evolutionary analyses, we identified the shallow pocket of the K13 KREP domain as a likely candidate surface for binding substrate molecule(s). Importantly, we observed that C580Y and R539T ART-R mutations cause local structural destabilization of the KREP structure rather than directly altering the shallow pocket. We also detected in the K13 BTB domain a conserved patch of sites that are involved in protein-protein interactions in known BTB-Cullin and BTB-BTB complexes. Efforts should now focus on the validation of the binding properties of the K13 KREP shallow pocket and identify its binding partner(s). This may help to clarify the structure-function relationship in K13.

To investigate the evolutionary regime that has shaped the k13 protein-coding DNA sequence during species evolution, we analyzed the non-synonymous (dN) to synonymous (dS) substitution rate ratio ω (=dN/dS), estimated by maximum-likelihood using the codeml tool from PAML v.4.834,69. ω provides a sensitive measure of selective pressure at the amino acid level by comparing substitution rates with statistical distribution and considering the phylogenetic tree topology. Typically, ω 1 indicate neutral evolution and positive selection respectively.

R.C., A.S. and J.C. designed the research. A.S. and J.C. supervised the whole work, and M.A.M. supervised the molecular dynamics simulations. R.C. performed the building of datasets, statistical and evolutionary analyses, structural visualization and molecular dynamics simulations. D.C.J. provided additional data and input on the research design. All the authors participated in interpretations. R.C., A.S. and J.C. wrote the first draft of the manuscript. All the authors contributed for editing the manuscript and approved the manuscript.

Collagen has a triple-stranded rope-like coiled structure. The major collagen of skin, tendon, and bone is the same protein containing 2 alpha-1 polypeptide chains and 1 alpha-2 chain. Although these are long (the procollagen chain has a molecular mass of about 120 kD, before the 'registration peptide' is cleaved off; see 225410), each messenger RNA is monocistronic (Lazarides and Lukens, 1971). Differences in the collagens from these 3 tissues are a function of the degree of hydroxylation of proline and lysine residues, aldehyde formation for cross-linking, and glycosylation. The alpha-1 chain of the collagen of cartilage and that of the collagen of basement membrane are determined by different structural genes. The collagen of cartilage contains only 1 type of polypeptide chain, alpha-1, and this is determined by a distinct locus. The fetus contains collagen of distinctive structure. The genes for types I, II, and III collagens, the interstitial collagens, exhibit an unusual and characteristic structure of a large number of relatively small exons (54 and 108 bp) at evolutionarily conserved positions along the length of the triple-helical gly-X-Y portion (Boedtker et al., 1983). The family of collagen proteins consists of a minimum of 9 types of collagen molecules whose constituent chains are encoded by a minimum of 17 genes (Ninomiya and Olsen, 1984).

Willing et al. (1993) pointed out that the abnormally low ratio of COL1A1 mRNA to COL1A2 (120160) mRNA in fibroblasts cultured from OI type I patients is an indication of a defect in the COL1A1 gene in the great majority of patients with this form of OI.

Whereas most cases of severe osteogenesis imperfecta result from mutations in the coding region of the COL1A1 or COL1A2 genes yielding an abnormal collagen alpha-chain, many patients with mild OI show evidence of a null allele due to a premature stop mutation in the mutant RNA transcript. As indicated in 120150.0046, mild OI in one case resulted from a null allele arising from a splice donor mutation where the transcript containing the included intron was sequestered in the nucleus. Nuclear sequestration precluded its translation and thus rendered the allele null. Using RT-PCR and SSCP of COL1A1 mRNA from patients with mild OI, Redford-Badwal et al. (1996) identified 3 patients with distinct null-producing mutations identified from the mutant transcript within the nuclear compartment. In a fourth patient with a gly-to-arg expressed point mutation, they found the mutant transcript in bot