A backbone-centred energy function of neural networks for protein design.

A protein backbone structure is designable if a substantial number of amino acid sequences exist that autonomously fold into it1,2. It has been suggested that the designability of backbones is governed mainly by side chain-independent or side chain type-insensitive molecular interactions3-5, indicating an approach for designing new backbones (ready for amino acid selection) based on continuous sampling and optimization of the backbone-centred energy surface. However, a sufficiently comprehensive and precise energy function has yet to be established for this purpose. Here we show that this goal is met by a statistical model named SCUBA (for Side Chain-Unknown Backbone Arrangement) that uses neural network-form energy terms. These terms are learned with a two-step approach that comprises kernel density estimation followed by neural network training and can analytically represent multidimensional, high-order correlations in known protein structures. We report the crystal structures of nine de novo proteins whose backbones were designed to high precision using SCUBA, four of which have novel, non-natural overall architectures. By eschewing use of fragments from existing protein structures, SCUBA-driven structure design facilitates far-reaching exploration of the designable backbone space, thus extending the novelty and diversity of the proteins amenable to de novo design.

Molecular basis for arginine C-terminal degron recognition by Cul2FEM1 E3 ligase.

Degrons are elements within protein substrates that mediate the interaction with specific degradation machineries to control proteolysis. Recently, a few classes of C-terminal degrons (C-degrons) that are recognized by dedicated cullin-RING ligases (CRLs) have been identified. Specifically, CRL2 using the related substrate adapters FEM1A/B/C was found to recognize C degrons ending with arginine (Arg/C-degron). Here, we uncover the molecular mechanism of Arg/C-degron recognition by solving a subset of structures of FEM1 proteins in complex with Arg/C-degron-bearing substrates. Our structural research, complemented by binding assays and global protein stability (GPS) analyses, demonstrates that FEM1A/C and FEM1B selectively target distinct classes of Arg/C-degrons. Overall, our study not only sheds light on the molecular mechanism underlying Arg/C-degron recognition for precise control of substrate turnover, but also provides valuable information for development of chemical probes for selectively regulating proteostasis.

Effects of ionic strength on the folding and stability of SAMP1, a ubiquitin-like halophilic protein.

Our knowledge of the folding behavior of proteins from extremophiles is limited at this time. These proteins may more closely resemble the primordial proteins selected in early evolution under extreme conditions. The small archaeal modifier protein 1 (SAMP1) studied in this report is an 87-residue protein with a ß-grasp fold found in the halophile Haloferax volcanii from the Dead Sea. To gain insight into the effects of salt on the stability and folding mechanism of SAMP1, we conducted equilibrium and kinetic folding experiments as a function of sodium chloride concentration. The results revealed that increasing ionic strength accelerates refolding and slows down unfolding of SAMP1, giving rise to a pronounced salt-induced stabilization. With increasing NaCl concentration, the rate of folding observed via a combination of continuous-flow (0.1-2 ms time range) and stopped-flow measurements (>2 ms) exhibited a >100-fold increase between 0.1 and 1.5 M NaCl and leveled off at higher concentrations. Using the Linderström-Lang smeared charge formalism to model electrostatic interactions in ground and transition states encountered during folding, we showed that the observed salt dependence is dominated by Debye-Hückel screening of electrostatic repulsion among numerous negatively charged residues. Comparisons are also drawn with three well-studied mesophilic members of the ß-grasp superfamily: protein G, protein L, and ubiquitin. Interestingly, the folding rate of SAMP1 in 3 M sodium chloride is comparable to that of protein G, ubiquitin, and protein L at lower ionic strength. The results indicate the important role of electrostatic interactions in protein folding and imply that proteins have evolved to minimize unfavorable charge-charge interactions under their specific native conditions.

Structural basis for METTL6-mediated m3C RNA methylation.

RNA modifications play important roles in mediating the biological functions of RNAs. 3-methylcytidine (m3C), albeit less abundant, is found to exist extensively in tRNAs, rRNAs and mRNAs. Human METTL6 is a m3C methyltransferase for tRNAs, including tRNASER(UGA). We solved the structure of human METTL6 in the presence of S-adenosyl-L-methionine and found by enzyme assay that recombinant human METTL6 is active towards tRNASER(UGA). Structural analysis indicated the detailed interactions between S-adenosyl-L-methionine and METTL6, and suggested potential tRNA binding region on the surface of METTL6. The structural research, complemented by biochemistry enzyme assay, will definitely shed light on the design of potent inhibitors for METTL6 in near future.

Crystal structure of TbAlba1 from Trypanosoma brucei.

Alba (Acetylation lowers binding affinity) domain is a small, dimeric nucleic acid-binding domain, which is widely distributed in archaea and numbers of eukaryotes. Alba domain containing proteins have been reported to be involved in many cellular processes, such as regulation of translation, maintaining genome stability, regulation of RNA processing and so on. In Trypanosoma brucei (T. brucei), there are four Alba proteins identified, which are named TbAlba1 to TbAlba4. However, the structure and function of TbAlba proteins are still unknown. Here, we solved the crystal structure of TbAlba1 to a resolution of 2.46 Å. TbAlba1 adopts a similar Alba-fold, which comprises of four ß-strands (ß1-ß4) and three long α-helices (α1-α3). Furthermore, TbAlba1 displays some structural features quite different from other Alba proteins. These differences may imply the diverse biological roles of Alba family members.

Molecular basis for cysteine oxidation by plant cysteine oxidases from Arabidopsis thaliana.

Plant Cysteine Oxidases (PCOs) play important roles in controlling the stability of Group VII ethylene response factors (ERF-VIIs) via Arg/N-degron pathway through catalyzing the oxidation of their N-Cys for subsequent Arginyl-tRNA--protein transferase 1 (ATE1) mediated arginine installation. Here we presented the crystal structures of PCO2, PCO4, and PCO5 from Arabidopsis thaliana (AtPCOs) and examined their in vitro activity by Mass spectrometry (MS). On the basis of Tris-bound AtPCO2, we modelled the structure of Cys-bound AtPCO2 and identified key AtPCO2 residues involved in N-Cys recognition and oxidation. Alanine substitution of potential N-Cys interaction residues impaired the activity of AtPCO5 remarkably. The structural research, complemented by mutagenesis and MS experiments, not only uncovers the substrate recognition and catalytic mode by AtPCOs, but also sheds light on the future design of potent inhibitors for plant cysteine oxidases.

Structural insights into SMCR8 C-degron recognition by FEM1B.

C-degrons play critical roles in targeting the receptor proteins of Cullin-RING E3 ligase complexes to initiate protein degradation. FEM1 proteins, including FEM1A, FEM1B, and FEM1C, act as the receptors to specifically recognize Arg/C-degrons to enable CRL2-mediated protein turnover. Very few substrates have been identified for FEM1B, except CDK5R1. We found that CRL2FEM1B also recognizes the C-degron of an SMCR8 isoform, and uncovered the recognition of SMCR8 by FEM1B through presenting the structure of FEM1B bound to SMCR8. Our work provides insights into the role of CRL2FEM1B in regulating the lifetime of SMCR8, a critical autophagy regulator.

Structural insight into HEMK2-TRMT112-mediated glutamine methylation.

Post-translational modifications play important roles in mediating protein functions in a wide variety of cellular events in vivo. HEMK2-TRMT112 heterodimer has been reported to be responsible for both histone lysine methylation and eukaryotic release factor 1 (eRF1) glutamine methylation. However, how HEMK2-TRMT112 complex recognizes and catalyzes eRF1 glutamine methylation is largely unknown. Here, we present two structures of HEMK2-TRMT112, with one bound to SAM and the other bound with SAH and methylglutamine (Qme). Structural analyses of the post-catalytic complex, complemented by mass spectrometry experiments, indicate that the HEMK2 utilizes a specific pocket to accommodate the substrate glutamine and catalyzes the subsequent methylation. Therefore, our work not only throws light on the protein glutamine methylation mechanism, but also reveals the dual activity of HEMK2 by catalyzing the methylation of both Lys and Gln residues.

Crystal structure of TbEsa1 presumed Tudor domain from Trypanosoma brucei.

The essential SAS2-related acetyltransferase 1 (Esa1), as a acetyltransferase of MYST family, is indispensable for the cell cycle and transcriptional regulation. The Tudor domain consists of 60 amino acids and belongs to the Royal family, which serves as a module interacting with methylated histone and/or DNA. Although Tudor domain has been widely studied in higher eukaryotes, its structure and function remain unclear in Trypanosoma brucei (T. brucei), a protozoan unicellular parasite causing sleeping sickness in human and nagana in cattle in sub-Saharan Africa. Here, we determined a high-resolution structure of TbEsa1 presumed Tudor domain from T. brucei by X-ray crystallography. TbEsa1 Tudor domain adopts a conserved Tudor-like fold, which is comprised of a five-stranded ß-barrel surrounded by two short α-helices. Furthermore, we revealed a non-specific DNA binding pattern of TbEsa1 Tudor domain. However, TbEsa1 Tudor domain showed no methyl-histone binding ability, due to the absence of key aromatic residues forming a conserved aromatic cage.

Solution structure of TbUfm1 from Trypanosoma brucei and its binding to TbUba5.

Ubiquitin-like proteins are conserved in eukaryotes and involved in numerous cellular processes. Ufm1 is proved to play important roles in endoplasmic reticulum homeostasis, vesicle transportation and embryonic development. Enzyme cascade of Ufm1 is similar to that of ubiquitin. Mature Ufm1 is activated and conjugated to substrates by assistance of Ufm1 activating enzyme Uba5 (E1), Ufm1 conjugating enzyme Ufc1 (E2), and Ufm1 ligating enzyme Ufl1 (E3). Here, we determined the solution structure of TbUfm1 from Trypanosoma brucei (T. brucei) by NMR spectroscopy and explored the interactions between TbUfm1 and TbUba5/TbUfc1/TbUfl1. TbUfm1 adopts a typical ß-grasp fold, which partially wraps a central α-helix and the other two helixes. NMR chemical shift perturbation indicated that TbUfm1 interacts with TbUba5 via a hydrophobic pocket formed by α1α2ß1ß2. Although the structure and Uba5-interaction mode of TbUfm1 are conserved in Ufm1 proteins, there are also some differences, which might reflect the potential diversity of Ufm1 in evolution and biological functions.

Solution structure of TbTFIIS2-2 PWWP domain from Trypanosoma brucei and its binding to H4K17me3 and H3K32me3.

Posttranslational modifications (PTMs) of core histones, such as histone methylation, play critical roles in a variety of biological processes including transcription regulation, chromatin condensation and DNA repair. In T. brucei, no domain recognizing methylated histone has been identified so far. TbTFIIS2-2, as a potential transcription elongation factors in T. brucei, contains a PWWP domain in the N-terminus which shares low sequence similarity compared with other PWWP domains and is absent from other TFIIS factors. In the present study, the solution structure of TbTFIIS2-2 PWWP domain was determined by NMR spectroscopy. TbTFIIS2-2 PWWP domain adopts a global fold containing a five-strand ß-barrel and two C-terminal α-helices similar to other PWWP domains. Moreover, through systematic screening, we revealed that TbTFIIS2-2 PWWP domain is able to bind H4K17me3 and H3K32me3. Meanwhile, we identified the critical residues responsible for the binding ability of TbTFIIS2-2 PWWP domain. The conserved cage formed by the aromatic amino acids in TbTFIIS2-2 PWWP domain is essential for its binding to methylated histones.

Structural basis for the recognition of kinesin family member 21A (KIF21A) by the ankyrin domains of KANK1 and KANK2 proteins.

A well-controlled microtubule organization is essential for intracellular transport, cytoskeleton maintenance, and cell development. KN motif and ankyrin repeat domain-containing protein 1 (KANK1), a member of KANK family, recruits kinesin family member 21A (KIF21A) to the cell cortex to control microtubule growth via its C-terminal ankyrin domain. However, how the KANK1 ankyrin domain recognizes KIF21A and whether other KANK proteins can also bind KIF21A remain unknown. Here, using a combination of structural, site-directed mutagenesis, and biochemical studies, we found that a stretch of ∼22 amino acids in KIF21A is sufficient for binding to KANK1 and its close homolog KANK2. We further solved the complex structure of the KIF21A peptide with either the KANK1 ankyrin domain or the KANK2 ankyrin domain. In each complex, KIF21A is recognized by two distinct pockets of the ankyrin domain and adopts helical conformations upon binding to the ankyrin domain. The elucidated KANK structures may advance our understanding of the role of KANK1 as a scaffolding molecule in controlling microtubule growth at the cell periphery.

Crystal structure of human YTHDC2 YTH domain.

N6-methyladenosine (m6A) "readers" play an important role in mRNA functions and metabolism. YTHDC2, as one of the m6A readers, controls fertileness through decreasing associated mRNA abundance and enhancing the translation efficiency of related mRNA via binding the targeted m6A RNA. However, how YTH domain of YTHDC2 recognize m6A RNA is still unknown. In this study, we determined the crystal structure of human YTHDC2 YTH domain, which adopts similar architecture to other solved YTH domain structures. YTHDC2 contains a conserved m6A binding pocket, and similar RNA binding surface shared by YTHDC1.

Solution structure of TbCentrin4 from Trypanosoma brucei and its interactions with Ca2+ and other centrins.

Centrin is a conserved calcium-binding protein that plays an important role in diverse cellular biological processes such as ciliogenesis, gene expression, DNA repair and signal transduction. In Trypanosoma brucei, TbCentrin4 is mainly localized in basal bodies and bi-lobe structure, and is involved in the processes coordinating karyokinesis and cytokinesis. In the present study, we solved the solution structure of TbCentrin4 using NMR (nuclear magnetic resonance) spectroscopy. TbCentrin4 contains four EF-hand motifs consisting of eight α-helices. Isothermal titration calorimetry experiment showed that TbCentrin4 has a strong Ca2+ binding ability. NMR chemical shift perturbation indicated that TbCentrin4 binds to Ca2+ through its C-terminal domain composed of EF-hand 3 and 4. Meanwhile, we revealed that TbCentrin4 undergoes a conformational change and self-assembly induced by high concentration of Ca2+ Intriguingly, localization of TbCentrin4 was dispersed or disappeared from basal bodies and the bi-lobe structure when the cells were treated with Ca2+in vivo, implying the influence of Ca2+ on the cellular functions of TbCentrin4. Besides, we observed the interactions between TbCentrin4 and other Tbcentrins and revealed that the interactions are Ca2+ dependent. Our findings provide a structural basis for better understanding the biological functions of TbCentrin4 in the relevant cellular processes.

The Protein Neddylation Pathway in Trypanosoma brucei: FUNCTIONAL CHARACTERIZATION AND SUBSTRATE IDENTIFICATION.

Protein posttranslational modifications such as neddylation play crucial roles in regulating protein function. Only a few neddylated substrates have been validated to date, and the role of neddylation remains poorly understood. Here, using Trypanosoma brucei as the model organism, we investigated the function and substrates of TbNedd8. TbNedd8 is distributed throughout the cytosol but enriched in the nucleus and the flagellum. Depletion of TbNedd8 by RNAi abolished global protein ubiquitination, caused DNA re-replication in postmitotic cells, impaired spindle assembly, and compromised the flagellum attachment zone filament, leading to flagellum detachment. Through affinity purification and mass spectrometry, we identified 70 TbNedd8-conjugated and -associated proteins, including known Nedd8-conjugated and -associated proteins, putative TbNedd8 conjugation system enzymes, proteins of diverse biological functions, and proteins of unknown function. Finally, we validated six Cullins as bona fide TbNedd8 substrates and identified the TbNedd8 conjugation site in three Cullins. This work lays the foundation for understanding the roles of protein neddylation in this early divergent parasitic protozoan.

Crystal structure of an ENT domain from Trypanosoma brucei.

Trypanosoma brucei (T. brucei) is a parasitic protozoan causing human sleeping sickness and related animal diseases. ENT (EMSY N-terminal) domain was first found in EMSY protein which has been proved to be involved in multiple biological processes such as DNA repair, tumorigenesis, and transcriptional regulation. So far, little is known about the function and structure of ENT domains from protozoan. Q385P5 from T. brucei, containing an ENT domain at its N-terminus, is a conserved protein in related kinetoplastid parasites. In this work, the crystal structure of ENT domain of Q385P5 (TbENT) was solved at a resolution of 2.3 Å. TbENT adopts a club-like shape consisting of five helixes, which is similar to the structure of human EMSY ENT domain (HsENT). Interestingly, TbENT shows significantly different orientation on the fifth α-helix compared with HsENT. Meanwhile, human HP1 interacts with a conserved motif adjacent to EMSY ENT domain. However, this conserved binding motif is absent in Q385P5. These differences may imply the different protein interactions and roles of Q385P5 and its ENT domain in T. brucei.

Recognition of hyperacetylated N-terminus of H2AZ by TbBDF2 from Trypanosoma brucei.

Histone modification plays an important role in various biological processes, including gene expression regulation. Bromodomain, as one of histone readers, recognizes specifically the ε-N-lysine acetylation (KAc) of histone. Although the bromodomains and histone acetylation sites of Trypanosoma brucei (T. brucei), a lethal parasite responsible for sleeping sickness in human and nagana in cattle, have been identified, how acetylated histones are recognized by bromodomains is still unknown. Here, the bromodomain factor 2 (TbBDF2) from T. brucei was identified to be located in the nucleolus and bind to the hyperacetylated N-terminus of H2AZ which dimerizes with H2BV. The bromodomain of TbBDF2 (TbBDF2-BD) displays a conserved fold that comprises a left-handed bundle of four α-helices (αZ, αA, αB, αC), linked by loop regions of variable length (ZA and BC loops), which form the KAc-binding pocket. NMR chemical shift perturbation further revealed that TbBDF2-BD binds to the hyperacetylated N-terminus of H2AZ through its KAc-binding pocket. By structure-based virtual screening combining with the ITC experiment, a small molecule compound, GSK2801, was shown to have high affinity to TbBDF2-BD. GSK2801 and the hyperacetylated N-terminus of H2AZ have similar binding sites on TbBDF2-BD. In addition, GSK2801 competitively inhibits the hyperacetylated N-terminus of H2AZ binding to TbBDF2-BD. After treatment of GSK2801, cell growth was inhibited and localization of TbBDF2 was disrupted. Our results report a novel bromodomain-histone recognition by TbBDF2-BD and imply that TbBDF2 may serve as a potential chemotherapeutic target for the treatment of trypanosomiasis.

Crystal structure of the WD40 domain of human PRPF19.

Human Pre-mRNA Processing factor 19 (hPRPF19) is an important component in human spliceosome machinery. hPRPF19 contains a WD40 repeats domain at its C-terminus, which is also conserved in yeast. Here we determined the crystal structure of the C-terminal WD40 repeat domain of hPRPF19 by X-ray crystallography. Our structural analysis revealed some significantly different structure features between the human and yeast Prp19 WD40 repeat domain. However, there are also conserved clusters of residues at the bottom surface of the fourth and the fifth WD40 repeats, which provides the important implication for the conserved Prp19 proteins in both human and yeast.

Solution structure of TbTFIIS2-1 PWWP domain from Trypanosoma brucei.

TbTFIIS2-1, one of the two TFIIS homologues of Trypanosome brucei (T. brucei), cooperates with TbTFIIS1 in regulating transcription in T. brcuei. Structurally divergent from other TFIIS homologues from higher organisms, TbTFIIS2-1 contains an additional N-terminal PWWP domain besides other three conserved domains, which may imply potential role of TbTFIIS2-1 in transcription regulation. Here, we determined the solution structure of PWWP domain of TbTFIIS2-1 by NMR spectroscopy, which was the first solution structure of PWWP domain solved in trypanosomatid. In spite of poor sequence similarity between PWWP domains, this domain of TbTFIIS2-1 adopts a conserved 3D-structure, which contains a five-stranded ß-barrel and a C-terminal α-helix. Furthermore, we found that TbTFIIS2-1 PWWP domain may be a protein-protein interaction module without the ability of DNA recognition and methyl-group interaction. Proteins 2016; 84:912-919. © 2016 Wiley Periodicals, Inc.

Structural basis for evolutionarily conserved interactions between TFIIS and Paf1C.

The elongation factor TFIIS interacts with Paf1C complex to facilitate processive transcription by Pol II. We here determined the crystal structure of the trypanosoma TFIIS LW domain in a complex with the LFG motif of Leo1, as well as the structures of apo-form TFIIS LW domains from trypanosoma, yeast and human. We revealed that all three TFIIS LW domains possess a conserved hydrophobic core that mediates their interactions with Leo1. Intriguingly, the structural study revealed that trypanosoma Leo1 binding induces the TFIIS LW domain to undergo a conformational change reflected in the length and orientation of α6 helix that is absent in the yeast and human counterparts. These differences explain the higher binding affinity of the TFIIS LW domain interacting with Leo1 in trypanosoma than in yeast and human, and indicate species-specific variations in the interactions. Importantly, the interactions between the TFIIS LW domain and an LFG motif of Leo1 were found to be critical for TFIIS to anchor the entire Paf1C complex. Thus, in addition to revealing a detailed structural basis for the TFIIS-Paf1C interaction, our studies also shed light on the origin and evolution of the roles of TFIIS and Paf1C complex in regulation of transcription elongation.