Argonaute-2 (4F3T) from Homo sapiens
Created by: Arianna Sherman
Argonaute proteins are critical components of the RNA-induced silencing complex (RISC), binding microRNA (miRNA) strands that guide the protein to its messenger RNA (mRNA) target (1). Argonaute proteins have been well described in prokaryotes, however, the function and associations of eukaryotic argonautes are poorly characterized (2). A member of the family of argonaute proteins, human Argonaute-2 (hAgo2) (PDB ID: 4F3T) has been found to bind tightly with short miRNA guide strands. The protein interacts with target mRNA through base pair complementarity and achieves RNA silencing through a relatively unknown "slicing" mechanism (2, 3).
The subunit structure of hAgo2 consists of four domains arranged into two lobes. The main domains of the protein are the N-terminal domain (NTD), the PIWI-Argonaute-Zwille (PAZ) domain, the middle (Mid) domain and the PIWI domain, which are joined together through structurally important linker sections (2). The molecular weight of hAgo2 is 97,208 Da and the isoelectric point is 9.32, as found from ExPASy, an online hub for bioinformatics resources (4).
hAgo2 is an essential component of the RISC that binds to miRNA guides and affects the silencing of target mRNA. miRNA is an inherently small single strand of RNA of about 22 base pairs numbered from 5' to 3'. The 5' end and 3' end of the strand will here on be referred to as 3' miRNA and 5' miRNA. miRNA guide strands regulate gene expression through translational inhibition or through direct cleavage of the target based on the degree of complementarity. Partial complementary interactions between the two RNA molecules result in translational repression, while perfect complementary interactions result in direct cleavage of mRNA (5). Argonaute proteins are important for viral defense mechanisms, and independent miRNA pathways function in the development of lymphoid and erythroid cells (5, 6). In argonaute-deficient D. melanogaster, susceptibility to viral infection was drastically increased from the argonaute-containing hosts, demonstrating the reliance of antiviral mechanisms on argonaute proteins. (6). Lymphoid and erythroid studies suggest that under-expression of Ago2 proteins cause defects in the development or function of these cells (5).
Four main domains characterize hAgo2: NTD, PAZ, Mid and PIWI. The structure and function of the NTD is relatively unknown, however, that the position of the domain is known to be based on binding of the target mRNA. There are linking sections between the NTD and the PAZ domain and between the PAZ domain and the Mid domain. While there is disagreement about whether these amino acid chains can be considered domains, they are important for the physical structure of the protein. Binding of target mRNA causes conformational changes in the linker chains and associated PAZ domain. Finally, the C-terminus of the protein is anchored in the miRNA binding groove through a water-mediated interaction with the 5' phosphate and steric interactions with Lys-566, Lys-570 and Arg-792 (3).
PAZ domain is the most distal of the four domains and is known to interact with the 3' miRNA guide (1, 2). The 3' miRNA is stabilized in this domain through hydrogen bonding interactions with His-271, His-316 and Tyr-311. In addition, an ionic bonding interaction between the 3' phosphate and Arg-315 helps to anchor 3' miRNA in the PAZ domain (3). This domain might also interact with a C-terminus insertion specific to eukaryotes that is responsible for two antiparallel alpha helices. These helices partially cover the binding groove, affecting the interaction between miRNA and target mRNA. This function of alpha helices in hAgo2 is one of the few functions known about the secondary structure of hAgo2, a structure consisting of mostly alpha helices and beta sheets (1). Finally, the PAZ domain is relatively flexible: the domain exhibits a range of orientations when binding to crystal lattice structures (3).
The Mid domain binds with a non-bridging oxygen atom of the 5' phosphate end of the miRNA guide (2). This oxygen atom interacts with Lys-566, which stabilizes the miRNA strand and neutralizes the charges on the phosphates of the 5' miRNA. At the binding site for the 5' miRNA, the Mid domain shows preference for uracil and adenine through a amino acid loop that has steric specificity (3).
The PIWI domain has been found to bind with non-bridging oxygen atoms on the 5' phosphate of the RNA guide strand, much like the Mid domain (1). The 5' uracil of the guide strand is bound to Tyr-529, resulting in the stabilization of the 5' miRNA of the guide by both the Mid and PIWI domains. The PIWI domain is also the site of target mRNA cleavage through a "slicing" mechanism. Upon binding, single cleavage slicing can occur if the interaction between the guide and the target is perfectly complimentary. If the match is not complementary, GW182 proteins, polyA-binding proteins (PABPC) and deadenylases are suspected to link the argonaute protein and its target (3). Phenol mediates interactions between proteins (e.g. GW182) and hAgo2. Any other function of phenol has not been elucidated (7).
One of the defining factors in the structure and function of argonaute proteins is their interaction with miRNA guide strands. hAgo2 is a poorly characterized, flexible protein that is stabilized by binding of the miRNA guide. The stability results from interactions between the miRNA strand and the binding groove that extend throughout all four domains. Binding of the miRNA guide locks the conformation (1).
The orientation of the miRNA guide along the binding groove is determined through interactions with critical residues throughout all four domains of the protein. Tyr-529 provides the foundation for the stacking of the 5' base of the miRNA guide and forms a hydrogen bond with the 5' phosphate. Lys-570, Arg-812 and the C-terminus of hAgo2 form water-mediated contacts with the 5' phosphate as well. These interactions anchor the 5' miRNA of the guide in the binding groove (7). The phosphates of bases 3-6 are stabilized by residues in the Mid and PIWI domains, while the phosphates of bases 7-9 interact with residues in the linker sections and the PIWI domain. Two important hydrogen bonds are formed between base 5 and the Tyr-804 and Ser-798 and between base 8 and the carbonyl of Ala-221. The aforementioned interactions result in the stabilization of the miRNA-hAgo2 interaction (1).
Kinks and turns in the backbone of the binding groove are important to the structure of the miRNA-hAgo2 complex (1). Three arginine residues form a large kink between bases 9 and 10 of the guide strand: Arg-710 stacks on base 9 and interacts with the phosphate of base 10, Arg-635 stabilizes base 10 (which also forms Van der Waals interactions with Ile-353), and Arg-351 interacts with the backbone phosphate of base 9. Proper placement of the miRNA strand results in proper placement of the mRNA scissile bond at the hAgo2 active site and correct orientation of the base-pairing interface of the guide strand (3). Alterations in these kinks and turns can result in misalignment of the miRNA and subsequent misdirection of the protein to the target mRNA strand (1).
Two servers were used to determine similarities between the protein of interest and potential comparison proteins. PSI-BLAST is a program that analyzes the primary structure of a given protein and compares the sequence with a database of sequences. Through analyzing the number of gaps in the sequence between two proteins, the program gives an "E value" for each comparison?the smaller the E value, the fewer gaps exist between primary structures (8). The Dali server identifies proteins with similar tertiary structure to a protein of interest. This identification is based on comparisons between the intermolecular distances of two proteins. The server denotes a "Z score" for each comparison?the higher the Z score, the more similar the tertiary structures of the two proteins. Dali only works for structures that contain an amino acid backbone, and therefore will only work for proteins, not for nucleic acids (9).
Human argonaute 1 (hAgo1) (PDB ID: 4KRE) was found to have structural similarities with hAgo2, being a member of the same family of proteins. This protein had an E score of 0.0 and a Z score of 61.1, indicating high similarity to hAgo2 in both its primary and tertiary structures (8, 9). hAgo1 is an inactive argonaute protein that does not silence messenger RNA, a functional change that most likely occurred from mutations in the sequence of the protein. Specifically, Pro-670 and Pro-675 were introduced in hAgo1, resulting in a kink near the inactive catalytic site in hAgo1 that prevents the complete binding of the guide strand to the binding groove. This alteration results in steric hindrance of the interaction between the guide strand and the target mRNA. Substitution of these proline residues with serine and glutamine resulted in an increase in catalytic activity, indicating that the kink formed by the proline residues in hAgo1 obstructs the miRNA binding groove. A possible method to regain catalytic activity in hAgo1 would be to cause the loop in the linker section of the protein (analogous to the linker section found in hAgo2) to move away from the binding groove, exposing more sites for base pairing. Another structural difference is the proximity of the NTD and PIWI domain. In hAgo1, there is a hydrophobic core that places these two domains close together; this core is missing in hAgo2 and the domains are more distal to each other.
There are several structural similarities between the hAgo1 and hAgo2 proteins. In both hAgo1 and hAgo2, the methods for 5' phosphate recognition are the same, and with the exception of the kink described above, the miRNA follows a similar trajectory in the binding groove of the proteins. In addition, the residues that constitute the binding groove of both proteins are completely conserved. (10).
Argonaute proteins are a critical part of the RISC, functioning through binding of miRNA to silence mRNA. The binding of miRNA in hAgo2 increases the stability of the protein through interactions that span all four characteristic domains. This stable form of the protein is structurally important for recognition of target strands and mechanisms of silencing mRNA (1). hAgo2 is biologically important as demonstrated by the role it plays in antiviral defense and the development of lymphoid and erythroid cells (5, 6). Despite the poor characterization of the Argonaute family in eukaryotes, it is clear that argonautes are essential to maintaining cellular processes.