Structural Insights into Protein-Aptamer Recognitions Emerged from Experimental and Computational Studies

Abstract

Aptamers are synthetic nucleic acids that are developed to target with high affinity and specificity chemical entities ranging from single ions to macromolecules and present a wide range of chemical and physical properties. Their ability to selectively bind proteins has made these compounds very attractive and versatile tools, in both basic and applied sciences, to such an extent that they are considered an appealing alternative to antibodies. Here, by exhaustively surveying the content of the Protein Data Bank (PDB), we review the structural aspects of the protein-aptamer recognition process. As a result of three decades of structural studies, we identified 144 PDB entries containing atomic-level information on protein-aptamer complexes. Interestingly, we found a remarkable increase in the number of determined structures in the last two years as a consequence of the effective application of the cryo-electron microscopy technique to these systems. In the present paper, particular attention is devoted to the articulated architectures that protein-aptamer complexes may exhibit. Moreover, the molecular mechanism of the binding process was analyzed by collecting all available information on the structural transitions that aptamers undergo, from their protein-unbound to the protein-bound state. The contribution of computational approaches in this area is also highlighted.

Keywords: NMR; X-ray crystallography; allostery; aptamer; cryo-electron microscopy; crystal structure; molecular dynamics; protein data bank; protein–aptamer interface; ternary complex.

Figure 1

Distribution of (a) the 144 PDB entries of protein–aptamer complexes reported since 1994 and (b) their resolution. Bars are colored according to the experimental technique used to determine the structure: X-ray crystallography (cyan), cryo-EM (green), and solution NMR (orange).

Figure 2

Correlation between interface areas and the number of H-bonding interactions for (a) the entire dataset of 152 protein–aptamer interfaces (R = 0.78, p < 10⁻⁵) and (b) the non-redundant ensemble including 67 interfaces (R = 0.78, p < 10⁻⁵). The entire dataset is colored (c) according to the experimental technique used to determine the structures (cyan for X-ray crystallography, green for cryo-EM, and orange for NMR) and (d) according to the chemical nature of the aptamer (green for DNA, red for RNA, and purple for NA-hybrid). In (d), complexes containing riboswitch aptamers and the complex of tRNA^Gln var-AGGU are represented with up-pointing triangle and plus symbols, respectively.

Figure 3

Correlation between interface areas and aptamer lengths for (a) the entire dataset of 152 interfaces (R = 0.14, p = 0.09). (b) Data are colored according to the chemical nature of the aptamer (green for DNA, red for RNA, and purple for NA-hybrid). In (b), complexes containing riboswitch aptamers and the complex of tRNA^Gln var-AGGU are represented with up-pointing triangle and plus symbols, respectively.

Figure 4

Selected examples of PDB structures endowed with a (a) small (entry #43, PDB ID 4M4O) and (b) large (entry #59, PDB ID 5D3G) protein–aptamer interface. (a) Lysozyme C and (b) HIV-1 reverse transcriptase are shown in blue. DNA and RNA aptamers are shown in green and red, respectively. Different shades of blue are used for the different protein chains of HIV-1 reverse transcriptase.

Figure 5

Selected examples of PDB structures of protein–aptamer complexes formed by monomeric proteins (blue): (a) von Willebrand factor (entry #16, PDB ID 3HXO), (b) coagulation factor Xa (entry #78, PDB ID 5VOE), (c) HIV-1 reverse transcriptase (entry #106, PDB ID 7OZ2), (d) double homeobox protein 4 (from left to right: entries #94 and 93, PDB ID 6U82 and 6U81), and (e) P16 peptide from a major prion protein (entry #40, PDB ID 2RU7). DNA and RNA aptamers are indicated in green and red, respectively. Different shades of the same color are used for the different protein/aptamer chains. For each structure, the protein–aptamer stoichiometry is indicated.

Figure 6

Selected examples of PDB structures of protein–aptamer ternary complexes. (a) Thrombin interacts with NU172 at exosite I and HD22_27mer at exosite II (entry #104, PDB ID 7NTU). (b) Two molecules of TBA-NNp/DDp bind the two thrombin exosites (entry #129, PDB ID 7ZKM). (c) RBD of SARS-CoV-2 spike protein S1 interacts with AM032-0 at the ACE2-binding site and AM047-0 at a distal site (entry #143, PDB ID 8J1Q). Different shades of the same color are used for the different protein/aptamer chains. In (c), the Fab domain of the imdevimab antibody is in light blue.

Figure 7

Selected examples of PDB structures of protein–aptamer complexes formed by homomeric proteins (blue): (a) insulin receptor (entry #118, PDB ID 7YQ3), (b) nerve growth factor (on the left, entry #58, PDB ID 4ZBN) and glutamate carboxypeptidase 2 (on the right, entry #89, PDB ID 6RTI), (c) lactate dehydrogenase (entry #70, PDB ID 5HRU), and (d) RNA-binding protein Hfq (from left to right: entries #20 and 21, PDB ID 3HSB and 3AHU). DNA and RNA aptamers are shown in green and red, respectively. Different shades of the same color are used for the different protein/aptamer chains. For each structure, the protein–aptamer stoichiometry is indicated.

Figure 8

Selected examples of PDB structures of protein–aptamer complexes in large protein assemblies: (a) MS2 coat protein (entry #6, PDB ID 5MSF) and (b) DNA-directed RNA polymerase (entry #136, PDB ID 8F3C). Proteins and RNA aptamers are shown in blue and red, respectively. DNA in (b) is shown in green. Different shades of the same color are used for the different protein/nucleic acid chains.

Figure 9

Structural superposition of the RNA aptamer targeting the ribosomal protein S8 (blue) in the protein-unbound (yellow, PDB ID 2LUN) and -bound (red, entry #53, PDB ID 4PDB) states.

Figure 10

Structural superposition of the RNA aptamer targeting NF-κB (blue) in the protein-unbound (yellow, PDB ID 2JWV) and -bound (red, entry #11, PDB ID 1OOA) states.

Figure 11

Structural superposition of the anti-prion RNA aptamer in the protein-unbound state (yellow, PDB ID 6K84) and bound to the P16 peptide from a major prion protein (blue), as reported in (a) entry #39 (red, PDB ID 2RSK) and (b) entry #40 (red, PDB ID 2RU7). Different shades of the same color are used for the different protein/aptamer chains.

Figure 12

Structural superposition of the theophylline RNA aptamer in the protein-unbound state (yellow, PDB ID 1O15) and bound to Fab BL3-6 (blue), as reported in (a) entry #134 (red, PDB ID 8DK7) and (b) entry #133 (red, PDB ID 8D29). The protein-bound aptamer in (b) does not bind theophylline.