Use of cyclic peptides to induce crystallization: case study with prolyl hydroxylase domain 2

Abstract

Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein–protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.

Structural basis for binding of the renal carcinoma target hypoxia‐inducible factor 2α to prolyl hydroxylase domain 2

“…substrate complexes, the β2-β3 loop folds to isolate the HIFα-ODD substrate proline at the active site. 11,[27][28][29][30] Overall, these observations support the proposal that β2-β3 loop dynamics are important both in catalysis and determining PHD/HIFα-ODD substrate selectivity, though the precise molecular details are undefined (Figure 1B,C). 27,29,30 VHL gene mutation is common in clear cell renal cell carcinoma (ccRCC) patients causing upregulation of HIFα isoforms, so increasing the expression of the HIF2α target VEGF, in a manner apparently promoting tumorigenesis and cancer progression.…”

“…Comparison of the overall PHD2 181‐426/407 .Mn.NOG/2OG.HIF2α‐CODD structures reveals conservation of the distorted double stranded β‐helix (DSBH) and associated HIFα‐ODD substrate binding elements (Figures 1B,C and 3). 11,27,28,36,37 The NOG and 2OG structures are very similar to each other (backbone RMSD: 0.078 Å) and, to a somewhat lesser extent in terms of details, with other PHD2.HIFα‐ODD structures (Figure 3). 11,27,28 In particular, variations in the conformations of α1, the β2–β3 loop, and the C‐terminal α4 regions are observed.…”

“…Note that the constructs used vary in the length of their C-terminus and in our case reversible binding of 3C may promote formation of, or stabilize, specific conformations that promote crystallization (Figure 3). 28 The previously reported PHD2 active site chemistry is also conserved in the PHD2 181-407 .Mn.NOG/2OG.HIF2α-CODD structures (Figure 3), 37 with a single manganese ion (substituting for iron) being coordinated by the side chains of His313, Asp315, and His374, as well as a well-defined water molecule/hydroxide ion. 11,27,28 The use of Mn(II) in PHD2 crystallization/inhibition is of interest given links between disease associated with Mn metabolism and erythropoiesis.…”

Structural Basis of Prolyl Hydroxylase Domain Inhibition by Molidustat

“… [23] Thus, at least to some extent, the precise nature of the available PHD structures likely reflects the varied conditions used for crystallisation (Table S3), which has been done using a range of ligands including both tight and weak binding inhibitors as well as substrate noncompetitive cyclic peptides binding away from the active site. [23] Nonetheless, it is of interest to compare our Molidustat/IOX4 structures with those of other triazole containing (IOX4‐A, and Takeda‐17) and non‐triazole‐containing inhibitors (e. g., Vadadustat and FG‐2216) with respect to interactions with Tyr303, Tyr310, Tyr329, and Arg383 (Figures 4 and S4). [ 9 , 24 ] Tyr303, Tyr310, and Arg383 are conserved in the animal PHDs and bacterial orthologues.…”

Advances in hybrid peptide-based self-assembly systems and their applications

“…Ordered forms of supramolecular structures of peptidic scaffolds can also be attained via crystallization. The hanging drop vapor diffusion method, 103,104 macromolecular crystallization of mesocrystals, 105 and slow evaporation method, 35,40,[106][107][108][109][110][111] are some techniques used for obtaining highly ordered crystalline structures (Fig. 5).…”