Yuan Chen

Small Ubiquitin-like Modifier (SUMO) Recognition of a SUMO Binding Motif A REVERSAL OF THE BOUND ORIENTATION

Sumoylation has recently been identified as an important mechanism that regulates protein interactions and localization in essential cellular functions, such as gene transcription, subnuclear structure formation, viral infection, and cell cycle progression. A SUMO binding amino acid sequence motif (SBM), which recognizes the SUMO moiety of modified proteins in sumoylation-dependent cellular functions, has been consistently identified by several recent studies. To understand the mechanism of SUMO recognition by the SBM, we have solved the solution structure of SUMO-1 in complex with a peptide containing the SBM derived from the protein PIASX (KVDVIDLTIESSSDEEEDPPAKR). Surprisingly, the structure reveals that the bound orientation of the SBM can reverse depending on the sequence context. The structure also reveals a novel mechanism of recognizing target sequences by a ubiquitin-like module. Unlike ubiquitin binding motifs, which all form helices and bind to the main β-sheet of ubiquitin, the SBM forms an extended structure that binds between the α-helix and a β-strand of SUMO-1. This study provides a clear mechanism of the SBM sequence variations and its recognition of the SUMO moiety in sumoylated proteins.

The Binding Interface between an E2 (UBC9) and a Ubiquitin Homologue (UBL1)

Human UBC9 is a member of the E2 (ubiquitin conjugation enzyme) family of proteins. Instead of conjugating to ubiquitin, it conjugates with a ubiquitin homologue UBL1 (also known as SUMO-1, GMP1, SMTP3, PIC1, and sentrin). UBC9 has been shown to be involved in cell cycle regulation, DNA repair, and p53-dependent processes. The binding interfaces of the UBC9 and UBL1 complex have been determined by chemical shift perturbation using nuclear magnetic resonance spectroscopy. The binding site of UBL1 resides on the ubiquitin domain, and the binding site of UBC9 is located on a structurally conserved region of E2 . Because the UBC9-UBL1 system shares many similarities with the ubiquitin system in structures and in conjugation with each other and with target proteins, the observed binding interfaces may be conserved in E2-ubiquitin interactions in general.

Repair of Methylation Damage in DNA and RNA by Mammalian AlkB Homologues

Human and Escherichia coli derivatives of AlkB enzymes remove methyl groups from 1-methyladenine and 3-methylcytosine in nucleic acids via an oxidative mechanism that releases the methyl group as formaldehyde. In this report, we demonstrate that the mouse homologues of the α-ketoglutarate Fe(II) oxygen-dependent enzymes mAbh2 and Abh3 have activities comparable to those of their human counterparts. The mAbh2 and mAbh3 release modified bases from both DNA and RNA. Comparison of the activities of the homogenous ABH2 and ABH3 enzymes demonstrate that these activities are shared by both sets of enzymes. An assay for the detection of α-ketoglutarate Fe(II) dioxygenase activity using an oligodeoxyribonucleotide with a unique modification shows activity for all four enzymes studied and a loss of activity for eight mutant proteins. Steady-state kinetics for removal of methyl groups from DNA substrates indicates that the reactions of the proteins are close to the diffusion limit. Moreover, mAbh2 or mAbh3 activity increases survival in a strain defective in alkB. The mRNAs of AHB2 and ABH3 are expressed most in testis for ABH2 and ABH3, whereas expression of the homologous mouse genes is different. The mAbh3 is strongly expressed in testis, whereas highest expression of mAbh2 is in heart. Other purified human AlkB homologue proteins ABH4, ABH6, and ABH7 do not manifest activity. The demonstration of mAbh2 and mAbh3 activities and their distributions provide data on these mammalian homologues of AlkB that can be used in animal studies.

Sequential Posttranslational Modifications Program FEN1 Degradation during Cell-Cycle Progression

We propose that cell-cycle-dependent timing of FEN1 nuclease activity is essential for cell-cycle progression and the maintenance of genome stability. After DNA replication is complete at the exit point of the S phase, removal of excess FEN1 may be crucial. Here, we report a mechanism that controls the programmed degradation of FEN1 via a sequential cascade of posttranslational modifications. We found that FEN1 phosphorylation stimulated its SUMOylation, which in turn stimulated its ubiquitination and ultimately led to its degradation via the proteasome pathway. Mutations or inhibitors that blocked the modification at any step in this pathway suppressed FEN1 degradation. Critically, the presence of SUMOylation- or ubiquitination-defective, nondegradable FEN1 mutant protein caused accumulation of Cyclin B, delays in the G1 and G2/M phases, and polyploidy. These findings may represent a newly identified regulatory mechanism used by cells to ensure precise cell-cycle progression and to prevent transformation.

A novel DNA-binding motif shares structural homology to DNA replication and repair nucleases and polymerases

A novel class of DNA-binding domains has been established from at least sixteen recently identified DNA-binding proteins. The three-dimensional structure of one of these domains, Mrf-2, has been solved using NMR methods. This structure is significantly different from known DNA-binding domain structures. The mechanism of DNA recognition by this motif has been suggested based on conserved residues, surface electrostatic potentials and chemical shift changes. This new DNA-binding motif shares structural homology with T4 RNase H, E. coli endonuclease III and Bacillus subtilis DNA polymerase I. The structural homology suggests a mechanism for substrate recognition by these enzymes.

Insights into high affinity small ubiquitin-like modifier (SUMO) recognition by SUMO-interacting motifs (SIMs) revealed by a combination of NMR and peptide array analysis.

Background: The SUMO-interacting motif (SIM) mediates SUMO-dependent regulation. Results: The structure of a SUMO1-specific SIM in complex with SUMO1 is solved, and the SIM sequence requirements are identified by peptide arrays. Conclusion: SIMs bound in the parallel orientation have more strictly conserved sequence than those in the antiparallel orientation. Significance: The findings will facilitate the identification of new SIMs and the design of SIM mimetics. The small ubiquitin-like modifiers (SUMOs) regulate many essential cellular functions. Only one type of SUMO-interacting motif (SIM) has been identified that can extend the β-sheet of SUMO as either a parallel or an antiparallel strand. The molecular determinants of the bound orientation and paralogue specificity of a SIM are unclear. To address this question, we have conducted structural studies of SUMO1 in complex with a SUMO1-specific SIM that binds to SUMO1 with high affinity without post-translational modifications using nuclear magnetic resonance methods. In addition, the SIM sequence requirements have been investigated by peptide arrays in comparison with another high affinity SIM that binds in the opposing orientation. We found that antiparallel binding SIMs tolerate more diverse sequences, whereas the parallel binding SIMs prefer the more strict sequences consisting of (I/V)DLT that have a preference in high affinity SUMO2 and -3 binding. Comparison of two high affinity SUMO1-binding SIMs that bind in opposing orientations has revealed common SUMO1-specific interactions needed for high affinity binding. This study has significantly advanced our understanding of the molecular determinants underlining SUMO-SIM recognition.

Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design

Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a “monobody,” a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with Kd values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

Sumoylation of SAE2 C Terminus Regulates SAE Nuclear Localization

Background: The mechanisms that regulate intracellular trafficking of the SUMOylation enzymes are not well understood. Results: SUMO modification of SAE2 at and near the NLS, in addition to its NLS, is required for its nuclear localization. Conclusion: A mechanism regulating SUMOylation activity in different cellular compartments was identified. Significance: This SUMOylation-dependent mechanism of regulating intracellular localization may occur widely. SUMOylation occurs predominantly in the nucleus, but non-nuclear proteins can also be SUMOylated. It is unclear how intracellular trafficking of the SUMOylation enzymes is regulated to catalyze SUMOylation in different cellular compartments. Here we report that the SAE2 subunit of human SUMO activation enzyme (SAE) underwent rapid nucleocytoplasmic shuttling and its nuclear accumulation depended on SUMO modification at the C terminus. The SUMOylation sites included three Lys residues on the bipartite nuclear localization sequence (NLS) and two Lys residues outside of but adjacent to the NLS, and their SUMOylation was catalyzed by Ubc9. Because SAE2 forms a tight heterodimer with SAE1 and it controls the trafficking of the heterodimer, this study has identified the mechanism used to localize SAE to the nucleus. Similar mechanisms are likely to exist for other proteins that depend on SUMOylation for nuclear localization.

Small Ubiquitin-like Modifier (SUMO) Modification of E1 Cys Domain Inhibits E1 Cys Domain Enzymatic Activity

Background: It has been unclear how E1 activities in ubiquitin-like modifications are regulated. Results: This study identified a role of SUMO modification of the Cys domain of the SUMO E1. Conclusion: The identified modification is a mechanism for “storing” a pool of E1 that can be quickly activated in response to environmental stress. Significance: Similar regulation likely exists across the homologous E1s of ubiquitin-like modifications. Although it is well established that ubiquitin-like modifications are tightly regulated, it has been unclear how their E1 activities are controlled. In this study, we found that the SAE2 subunit of the small ubiquitin-like modifier (SUMO) E1 is autoSUMOylated at residue Lys-236, and SUMOylation was catalyzed by Ubc9 at several additional Lys residues surrounding the catalytic Cys-173 of SAE2. AutoSUMOylation of SAE2 did not affect SUMO adenylation or formation of E1·SUMO thioester, but did significantly inhibit the transfer of SUMO from E1 to E2 and overall SUMO conjugations to target proteins due to the altered interaction between E1 and E2. Upon heat shock, SUMOylation of SAE2 was reduced, which corresponded with an increase in global SUMOylation, suggesting that SUMOylation of the Cys domain of SAE2 is a mechanism for “storing” a pool of E1 that can be quickly activated in response to environmental changes. This study is the first to show how E1 activity is controlled by post-translational modifications, and similar regulation likely exists across the homologous E1s of ubiquitin-like modifications.

Conformational Transition Associated with E1-E2 Interaction in Small Ubiquitin-like Modifications

Ubiquitin-like modifications regulate nearly every aspect of cellular functions. A key step in these modifications is the recognition of the carrier enzyme (E2) by the activating enzyme (E1). In this study, we have found that a critical E2-binding surface on the E1 of the small ubiquitin-like modifier has unusually high populations in both ordered and disordered states. Upon binding the E2, the disordered state is converted to the ordered state, which resembles the structure of the bound conformation, providing a mechanism to resolve the “Levinthal Paradox” search problem in a folding-upon-binding process. The significance of the folding-unfolding equilibrium is shown by the loss of functions of the mutations that shift the equilibrium to the folded state. This study highlights the importance of conformational flexibility in the molecular recognition event.