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Abundant Perithecial Protein (APP) consists of a domain, containing an Ig-like domain and is commonly found in perithecia structures found in plants and fungi, where it resembles photosynthetic processes. APP is deficient in one Ca2+ binding property, similar to its homolog DdCAD-1, a calcium-binding cell-adhesion molecule. Because APP has dispensed it’s Ca2+ binding properties in exchange for increased stability, researchers are led to believe that APP is an ancestor of ocular crystallins due to its native crystallin-like attributes. Both 5Z6D and 5Z6E play key roles in cell adhesion. APP has been identified in neurospora crassa, Sordaria macrospora, two other members of Xylariales, and one species of aspergillus. APP is mostly found in fungi, maintaining high levels of sequence variance.

5Z6D

presents as a DUF1881 domain-containing, double-chain beta gamma-crystallin structure derived from APP, found within neurospora crassa. This chain has been identified with a 1.60 Å resolution. Though resolution is considered above average, the Rfree is calculated 0.226, considered below average when compared to the resolution value. Chain A contains 215 proteins coded within its sequence. Chain B has been crystallized, and found to maintain the same sequence. With a total of 408 amino acids in the protein, it is nearly 4.5 times larger than 5Z6E and has over double the protein length, at 47.8 kDa. Unlike 5Z6E, this variant has zero ligands in its structure. The complete 3D guided tour can be found here. A basic review of statistical crystallography information can be found in PDBj.

5Z6E

is a single-chain, beta gamma-crystallin structure of APP. FASTA format of amino acid sequence of 5Z6E can be found here. Full length genomic chains and be observed in UniProt. Its complete structure and 3D analysis can be located in the OCA atlas. A summary of statistical data may be found here. For a complete 3D guided tour, FirstGlance is recommended. With a resolution of 1.86 Å, the resolution is stronger than that of 5Z6D, Rfree is calculated to be 0.201, presenting greater reliability when considering the resolution. Containing 90 amino acids in its sequence, 5Z6E maintains a length of 101 proteins at only 11.2 kDa. Unlike 5Z6D, there are 2 identified ligands; CA and K are located at D39, S81, Q56, and A48, A50, D88, V46, and T68, respectively.

Heterodomain

APP and DdCAD-1 exist within a specific heterodomain, exemplified by the binding interactions between the Ig-like domain and the N-terminus beta gamma-crystallin domain. This genomic organization has been observed across nearly 100 protein sequences found within NCBI, as represented by gamma-proteobacteria. These proteins are limited to fungi and slime molds when contained to eukaryotes. APP has been found to contain 30% sequence similarity when compared to that of DdCAD-1, which also exists within slime molds. When expressed, DdCAD-1 resembles a cytosolic soluble protein as transported via vacuoles across the plasma membrane. DdCAD-1’s interactions with Ca2+ presents the possibility of involvement with homeostasis, where DdCAD-1 has 3 Ca2+ binding sites, two of which exist within the beta gamma-crystallin domain, and one between the Ig-like domain and beta gamma-crystallin domain. Other various studies suggest DdCAD-1 may assist in cell differentiation, as well as cell proportioning. While DdCAD-1 binds with Ca2+, APP does not, despite its functional Ca2+ binding site. 3D structures of DdCAD-1, both 1YHP and 2B1O can be viewed on FirstGlance.

Evolutionary Relevance

When interacted with an Ig-like domain, one of two Ca2+ binding sites will render inoperable. With one degenerated protein, and one functional protein, 5Z6E resembles an evolutionary intermediate of ocular crystallins. Ca2+ binding sites will interact with APP N-terminus domain (APP-NTD), revealing interface interactions and render the Ca2+ binding site nonfunctional. Although APP-NTD binds with a micromolar affinity that is similar to DdCAD-1, it does not genuinely bond with Ca2+ binding sites as found on 5Z6E. Such findings of interface interaction between APP-NTD and the Ca2+-binding sites reveals a novel approach of Ca2+ binding regulation with respect to APP. Mutating surface amino acids, or separating from the Ig-like domain will alter structural constraints necessary for conformational transitions, allowing the beta gamma-crystallin domain to interact with Ca2+ binding sites. The correlation between increased stability, associated with the loss of Ca2+ binding is observed in both lens gamma-crystallins and APP. Mutant APP which allows for restoration of Ca2+ is less stable than the wild type which corresponds to the APP that does not bind to Ca2+. Overall, the correlation between the gain and loss of Ca2+ as observed in APP is additionally present in beta gamma-crystallins. These similarities are significant to the correlations between crystalline and noncrystalline propensities of both APP and DdCAD-1. The ability to maintain structure as a protein is paramount to the ability to evolve, making the protein less likely to alter their wild type structure, due to their native stability.

Ca2+ Binding Domain of APP-NTD

Both the resolution, at 1.86 Å and it’s crystallized structure reveal a beta gamma-crytallin fold within APP-NTD. Here, there has only been one identified density for Ca2+. As the Ca2+ observed in studies consists of 4 amino acid residues and 3 water molecules, the conformation presents a bipyramidal pentagon. As such, the main carbonyl of Gln56 from the beta hairpin interacts with Gly79 from the second loop. Asp 39 interacts with Ser81 by the side chain oxygen. The remaining coordinations of Ca2+ are filled by existing water molecules.

Ca2+ coordination of APP-NTD is homogenous to the canonical Ca2+-binding beta gamma-crystallins, though Serine and Thymine are replaced with Phe42 in residues 38-43 (NDKYFS) in the first Ca2+ binding site. The observed change is undesirable by Ca2+. Combined with the occupancy of Gly78 in the IGGLSR within the second greek motif, further side-chain coordination of Ca2+ and the N/D-N/D-X1-X2-S/T-S motif is undesired.

APP-NTD is best identified by its Y/FXXXXY/FG sequence within the beta gamma hairpin of the beta gamma-crystallin domain, stabilized by serine. The first greek motif sequence, YQKKNFEG and NDKYFS (residues 13-20, and 38-43, respectively), along with the beta-hairpin conforms similar to that of beta-hairpins found in similar beta gamma-crystallins. Alternatively, the second greek motif of APP-NTD has been found to reveal great variance in these sequences, amongst WQHYNETG and IGGLSR (residues 55-62 and 77-82, respectively). Regardless of the variations, residues 55-62 continue to envelope in a beta-hairpin. Studies show this hairpin is supported by Glu60, as it coordinates with Arg82 in residues 38-43 to stabilize the structure. Overall, findings show that APP-NTD presents a functioning and degenerated Ca2+ binding site, where the functional site coordinates with the second Ca2+-binding site of other similar binding sites found within beta gamma-crystallins. This class of proteins, thus, can only bind to one Ca2+ at the second binding site, seeing as the first binding site is disabled.

Ca2+ Binding Domain of APP

Alternatively, APP does not genuinely bind with Ca2+, though APP-TD does. If Ca2+ is capable of binding to APP at all, it is at least not within quantifiable range as measured by limits of ITC. This is likely due to the thermal unfolding behaviors when exposed to EDTA and Ca2+,which is comparable to a two-state transition. Here, approximate TM values are 69.6 and 70.2 C, respectively. Further experiments, such as DSC, further confirm that AP does not present thermal changes when exposed to Ca2+, implying further that no appreciable bond is formed at the binding site. Additionally, the Dmax value is 71 Å, and does not vary in the presence of Ca2+, further suggesting no changes to APP. NSD values of EDTA and APP are 1.37, where NSD value of Ca2+ is a calculated 1.42, implying further that solution scattering studies do not reflect Ca2+ binding. The two domain structure of APP consists of the N-terminal domain, being a beta gamma-crystallin, and the Ig-like domain located at the C-terminus. Each domain interacts with the other to form a tightly coiled protein. Likely, APP is not able to bind to Ca2+ due to the near-identical structure of half the N-terminus and the core of each domain. The Ca2+ coordinating residues of APP will reconform into loops 1 and 2 in the second greek motif, N/D-N/D-X1-X2-ST/S motif and the beta hairpin. Met121 is localized to the surface of APP, though it does not play a role in major interactions of APP, except for the hydrogen bonds which mediate Glu60 of the main carbon chain. APP does not provide Ca2+ densities in crystallization, though crystals have been identified in the presence of 10-30mM CaCl2. Overall, APP fails to provide statistics associated with Ca2+. In APP, the primary carbonyl oxygen, in the second hairpin, of Gln56 coordinates the positive x-position in APP-NTD is relocated from 6.7 Å in APP-NTD and rotate toward the Ig-like domain, and away from the alternative Ca2+ binding site. Likewise, the hydroxyl group of Ser81 relocated 3.5 Å further from the Ca2+ binding site of APP-NTD. Further, the orientation of Gln56 primary chain and the side chain of Ser81 as located in APP will degenerate the APP Ca2+ binding site. Further, Asp39 coordinates with the negative x-position, and also shifts 3.1 Å. This distance spanning the two molecules is 14 Å with respect to APP, and is 4.7 Å when APP-NTD is bound with Ca2+. The exaggerated shifts in distance between the two molecules is significant, considering the radius of gyration (Rg) of 11.8 combined with a particle dimension (Dmax) of 40.2 Å. Overall, the 3D structure of APP is largely culprit of its inability to bind Ca2+, where the residues residing within the Ca2+ binding site in APP-NTD are unalike those of APP, further promoting the disability of the Ca2+ binding site of the parent APP.

References

Asmita D. Pawar, Uday Kiran, Rajeev Raman, Sushil Chandani, Yogendra Sharma, Abundant Perithecial Protein (APP) from Neurospora is a primitive functional analog of ocular crystallins. Biochemical and Biophysical Research Communications. Volume 516, Issue 3, 2019. Pages 796-800. ISSN 0006-291X. https://doi.org/10.1016/j.bbrc.2019.06.102.

Swaroop Srivastava, S., Raman, R., Kiran, U., Garg, R., Chadalawada, S., Pawar, A.D., Sankaranarayanan, R. and Sharma, Y. (2018), Interface interactions between βγ-crystallin domain and Ig-like domain render Ca2+-binding site inoperative in abundant perithecial protein of Neurospora crassa. Mol Microbiol, 110: 955-972. https://doi.org/10.1111/mmi.14130