Structure-function study of maize ribosome-inactivating protein: implications for the internal inactivation region and the sole glutamate in the active site

Maize ribosome-inactivating protein is classified as a class III or an atypical RNA N-glycosidase. It is synthesized as an inactive precursor with a 25-amino acid internal inactivation region, which is removed in the active form. As the first structural example of this class of proteins, crystals of the precursor and the active form were diffracted to 2.4 and 2.5 Å, respectively. The two proteins are similar, with main chain root mean square deviation (RMSD) of 0.519. In the precursor, the inactivation region is found on the protein surface and consists of a flexible loop followed by a long α-helix. This region diminished both the interaction with ribosome and cytotoxicity, but not cellular uptake. Like bacterial ribosome-inactivating proteins, maize ribosome-inactivating protein does not have a back-up glutamate in the active site, which helps the protein to retain some activity if the catalytic glutamate is mutated. The structure reveals that the active site is too small to accommodate two glutamate residues. Our structure suggests that maize ribosome-inactivating protein may represent an intermediate product in the evolution of ribosome-inactivating proteins. © 2007 The Author(s).published_or_final_versio

A switch-on mechanism to activate maize ribosome-inactivating protein for targeting HIV-infected cells

Maize ribosome-inactivating protein (RIP) is a plant toxin that inactivates eukaryotic ribosomes by depurinating a specific adenine residue at the α-sarcin/ricin loop of 28S rRNA. Maize RIP is first produced as a proenzyme with a 25-amino acid internal inactivation region on the protein surface. During germination, proteolytic removal of this internal inactivation region generates the active heterodimeric maize RIP with full N-glycosidase activity. This naturally occurring switch-on mechanism provides an opportunity for targeting the cytotoxin to pathogen-infected cells. Here, we report the addition of HIV-1 protease recognition sequences to the internal inactivation region and the activation of the maize RIP variants by HIV-1 protease in vitro and in HIV-infected cells. Among the variants generated, two were cleaved efficiently by HIV-1 protease. The HIV-1 protease-activated variants showed enhanced N-glycosidase activity in vivo as compared to their un-activated counterparts. They also possessed potent inhibitory effect on p24 antigen production in human T cells infected by two HIV-1 strains. This switch-on strategy for activating the enzymatic activity of maize RIP in target cells provides a platform for combating pathogens with a specific protease

The C-terminal fragment of the ribosomal P protein complexed to trichosanthin reveals the interaction between the ribosome-inactivating protein and the ribosome

Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by enzymatically depurinating a specific adenine residue at the sarcin-ricin loop of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation centre of the ribosome. Here, we present the 2.2 Å crystal structure of trichosanthin (TCS) complexed to the peptide SDDDMGFGLFD, which corresponds to the conserved C-terminal elongation factor binding domain of the ribosomal P protein. The N-terminal region of this peptide interacts with Lys173, Arg174 and Lys177 in TCS, while the C-terminal region is inserted into a hydrophobic pocket. The interaction with the P protein contributes to the ribosome-inactivating activity of TCS. This 11-mer C-terminal P peptide can be docked with selected important plant and bacterial RIPs, indicating that a similar interaction may also occur with other RIPs

Maize ribosome-inactivating protein uses Lys158-lys161 to interact with ribosomal protein P2 and the strength of interaction is correlated to the biological activities.

Ribosome-inactivating proteins (RIPs) inactivate prokaryotic or eukaryotic ribosomes by removing a single adenine in the large ribosomal RNA. Here we show maize RIP (MOD), an atypical RIP with an internal inactivation loop, interacts with the ribosomal stalk protein P2 via Lys158-Lys161, which is located in the N-terminal domain and at the base of its internal loop. Due to subtle differences in the structure of maize RIP, hydrophobic interaction with the 'FGLFD' motif of P2 is not as evidenced in MOD-P2 interaction. As a result, interaction of P2 with MOD was weaker than those with trichosanthin and shiga toxin A as reflected by the dissociation constants (K(D)) of their interaction, which are 1037.50 ± 65.75 µM, 611.70 ± 28.13 µM and 194.84 ± 9.47 µM respectively.Despite MOD and TCS target at the same ribosomal protein P2, MOD was found 48 and 10 folds less potent than trichosanthin in ribosome depurination and cytotoxicity to 293T cells respectively, implicating the strength of interaction between RIPs and ribosomal proteins is important for the biological activity of RIPs. Our work illustrates the flexibility on the docking of RIPs on ribosomal proteins for targeting the sarcin-ricin loop and the importance of protein-protein interaction for ribosome-inactivating activity

Engineering an Enhanced EGFR Engager: Humanization of Cetuximab for Improved Developability

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase whose proliferative effects can contribute to the development of many types of solid tumors when overexpressed. For this reason, EGFR inhibitors such as cetuximab can play an important role in treating cancers such as colorectal cancer and head and neck cancer. Cetuximab is a chimeric monoclonal antibody containing mouse variable regions that bind to EGFR and prevent it from signaling. Although cetuximab has been used clinically since 2004 to successfully control solid tumors, advances in protein engineering have created the opportunity to address some of its shortcomings. In particular, the presence of mouse sequences could contribute to immunogenicity in the form of anti-cetuximab antibodies, and an occupied glycosylation site in FR3 can contribute to hypersensitivity reactions and product heterogeneity. Using simple framework graft or sequence-/structure-guided approaches, cetuximab was humanized onto 11 new frameworks. In addition to increasing humanness and removing the VH glycosylation site, dynamic light scattering revealed increases in stability, and bio-layer interferometry confirmed minimal changes in binding affinity, with patterns emerging across the humanization method. This work demonstrates the potential to improve the biophysical and clinical properties of first-generation protein therapeutics and highlights the advantages of computationally guided engineering

MOD can be crosslinked to rat liver ribosome and P2.

<p>Crosslinking reactions were carried with individual proteins (lanes 1 and 3, 4 and 6) or mixtures of two proteins (lanes 2 and 5) and subject for western analysis. Protein bands were detected by anti-myc, anti-MOD and anti-P antibodies.</p

Interaction between RIPs and P2.

<p><b>A) Interaction of MOD, TCS and StxA with P2 at different ionic strengths.</b> Pull-down assay was conducted on RIPs under various ionic strengths to compare their strength of interaction with P2. Input indicates the same amount of purified RIPs was loaded to the P2-sepharose column and proteins were eluted using buffer with 1M NaCl. <b>B) Interactions between RIPs and C-terminal truncated P2.</b> C-terminal truncated P2 variants were subject to pull down assay with RIPs. The C-terminal amino acid sequences of P2, P2 [ΔC5] and P2 [ΔC10] are: AEEKKDEKKEESEE<b>SDDDMGFGLFD</b>, AEEKKDEKKEESEE<b>SDDDMG</b> and AEEKKDEKKEESEE<b>S</b> respectively. The bold letters refer to the conserved residues in P-proteins.</p

Relative <i>N</i>-glycosidase activities of maize RIP and its variants on 28S rRNA and rat liver ribosome.

<p>Relative <i>N</i>-glycosidase activities of maize RIP and its variants on 28S rRNA and rat liver ribosome.</p

Residues on MOD that correspond to the C11-P interacting residues on TCS.

<p><b>A) Superimposed structures of MOD (pdb: 2PQI) and TCS-C11-P complex (pdb: 2JDL).</b> The beta strands β7 and β8 in TCS (wheat) are replaced by the helix αI in MOD (cyan) while helices αG and αJ are conserved. P2-binding residues on MOD and TCS are distant apart as indicated in red and green respectively. <b>B) Comparison of C11-P interacting residues on TCS and the corresponding residues on MOD.</b> Structures of MOD and TCS are superimposed to locate the residues on MOD (colored in cyan and labelled in black and italic) corresponding to the C11-P interacting residues on TCS (colored in wheat and labelled in red). Many residues in these two RIPs are different, especially those at the hydrophobic patch of TCS (Phe-166, Val-223, Ile-225, Gly-231, Val-232 and Asn-236), suggesting MOD may interact with C11-P at a different site. <b>C) Stereo image zooming in the hydrophobic pocket of MOD (colored in cyan) with C11-P (colored in yellow).</b> The model reveals Arg-275 on MOD may crash with Leu-9 on C11-P while Glu-272 confronts directly Asp-11. Residues on TCS that interact with C11-P are highlighted in wheat for reference.</p

Screening of basic residues on MOD that are responsible for ribosome binding.

<p>The indicated residues were mutated to alanine and screened for their abilities to bind ribosome. W and E denote last wash and elution respectively.</p