X‐ray structures of Sap1 and Sap5: Structural comparison of the secreted aspartic proteinases from Candida albicans

Abstract

Proteolytic activity is an important virulence factor for Candida albicans (C. albicans). It is attributed to the family of the secreted aspartic proteinases (Saps) from C. albicans with a minimum of 10 members. Saps show controlled expression and regulation for the individual stages of the infection process. Distinct isoenzymes can be responsible for adherence and tissue damage of local infections, while others cause systemic diseases. Earlier, only the structures of Sap2 and Sap3 were known. In our research, we have now succeeded in solving the X-ray crystal structures of the apoenzyme of Sap1 and Sap5 in complex with pepstatin A at 2.05 and 2.5 A resolution, respectively. With the structure of Sap1, we have completed the set of structures of isoenzyme subgroup Sap1-3. Of subgroup Sap4-6, the structure of the enzyme Sap5 is the first structure that has been described up to now. This facilitates comparison of structural details as well as inhibitor binding modes among the different subgroup members. Structural analysis reveals a highly conserved overall secondary structure of Sap1-3 and Sap5. However, Sap5 clearly differs from Sap1-3 by its electrostatic overall charge as well as through structural conformation of its entrance to the active site cleft. Design of inhibitors specific for Sap5 should concentrate on the S4 and S3 pockets, which significantly differ from Sap1-3 in size and electrostatic charge. Both Sap1 and Sap5 seem to play a major part in superficial Candida infections. Determination of the isoenzymes' structures can contribute to the development of new Sap-specific inhibitors for the treatment of superficial infections with a structure-based drug design program.

Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

“…The crystal structure of Sap5 has been solved (37) and extrapolated to the protein structure for the Sap4 to -6 subgroup. There is a highly conserved secondary structure of the middle and back regions of Sap5 that contains the aspartic proteinase active-site cleft required for substrate and inhibitor (pepstatin A) binding (37). There are also three extended loop structures (arms 1, 2, and 3) surrounding this cleft, among which the arm 1 loops of Sap4 to -6 contain at least one integrin-binding motif (RGD) at the surface-exposed tip of the loop (29).…”

The Inflammatory Response Induced by Aspartic Proteases of Candida albicans Is Independent of Proteolytic Activity

“…The association of rSaps with human monocytes was analyzed by flow cytometry. Monocytes were incubated with different doses of FITC-conjugated rSaps and CTLA-4 F(ab) 2 for different periods at 37°C in the presence of 5% CO 2 . After incubation, cells were collected by centrifugation, fixed in 1% paraformaldehyde in phosphate-buffered saline (PBS), washed twice, and resuspended in PBS containing 0.5% bovine serum albumin and 0.1% sodium azide.…”

“…Individual Sap proteases have been purified from C. albicans culture supernatants (18) or by recombinant expression in Pichia pastoris (3) or Escherichia coli (24,25), and their biochemical properties have been characterized (2,3,55). For example, the optimum pHs for individual Saps have been established as between pH 2.0 and 5.5 for Saps 1 to 3 and between pH 3.0 and 7.0 for Saps 4 to 6 (3).…”

HIV aspartyl protease inhibitors as promising compounds againstCandida albicansAndré Luis Souza dos Santos

“…In this context, the crystal structure of Sap2 complexed with pepstatin A has been known since 1993 [107] , whereas the crystal structure of Sap3 and its complex with pepstatin A was first presented in 2007 [108] . The secondary structures of Sap2 and Sap3 as well as Sap1 and Sap5 were recently described [109] . These data could help in the development of novel and more effective anti-C. albicans compounds.…”