Gary W. Daughdrill

Classification of Intrinsically Disordered Regions and Proteins.

1.1. Uncharacterized Protein Segments Are a Source of Functional Novelty Over the past decade, we have observed a massive increase in the amount of information describing protein sequences from a variety of organisms.1,2 While this may reflect the diversity in sequence space, and possibly also in function space,3 a large proportion of the sequences lacks any useful function annotation.4,5 Often these sequences are annotated as putative or hypothetical proteins, and for the majority their functions still remain unknown.6,7 Suggestions about potential protein function, primarily molecular function, often come from computational analysis of their sequences. For instance, homology detection allows for the transfer of information from well-characterized protein segments to those with similar sequences that lack annotation of molecular function.8−10 Other aspects of function, such as the biological processes proteins participate in, may come from genetic- and disease-association studies, expression and interaction network data, and comparative genomics approaches that investigate genomic context.11−17 Characterization of unannotated and uncharacterized protein segments is expected to lead to the discovery of novel functions as well as provide important insights into existing biological processes. In addition, it is likely to shed new light on molecular mechanisms of diseases that are not yet fully understood. Thus, uncharacterized protein segments are likely to be a large source of functional novelty relevant for discovering new biology.

Evolution and disorder

The evolution of disordered proteins or regions of proteins differs from that of ordered proteins because of the differences in their sequence composition, intramolecular contacts, and function. Recent assessments of disordered protein evolution at the sequence, structural, and functional levels support this hypothesis. Disordered proteins have a different pattern of accepted point mutations, exhibit higher rates of insertions and deletions, and generally, but not always, evolve more rapidly than ordered proteins. Even with these high rates of sequence evolution, a few examples have shown that disordered proteins maintain their flexibility under physiological conditions, and it is hypothesized that they maintain specific structural ensembles.

Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal Degradation

Background: There is an urgent need to develop small molecule Mcl-1-specific inhibitors for the treatment of Mcl-1-dependent ABT-737/263-resistant cancers. Results: Maritoclax binds to and induces Mcl-1 degradation, thereby leading to Mcl-1-dependent apoptosis and sensitizing leukemia/lymphoma cells to ABT-737. Conclusion: Maritoclax is a novel Mcl-1-specific inhibitor. Significance: Antagonizing Mcl-1 by maritoclax has the potential to prevent and overcome Mcl-1-mediated resistance to ABT-737/263. The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-XL and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-XL with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-XL, and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-XL-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1–2 μm. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.

The alphabet of intrinsic disorder

A significant fraction of every proteome is occupied by biologically active proteins that do not form unique three-dimensional structures. These intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) have essential biological functions and are characterized by extensive structural plasticity. Such structural and functional behavior is encoded in the amino acid sequences of IDPs/IDPRs, which are enriched in disorder-promoting residues and depleted in order-promoting residues. In fact, amino acid residues can be arranged according to their disorder-promoting tendency to form an alphabet of intrinsic disorder that defines the structural complexity and diversity of IDPs/IDPRs. This review is the first in a series of publications dedicated to the roles that different amino acid residues play in defining the phenomenon of protein intrinsic disorder. We start with proline because data suggests that of the 20 common amino acid residues, this one is the most disorder-promoting.

NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain

The interaction between the acidic transactivation domain of the human tumor suppressor protein p53 (p53TAD) and the 70 kDa subunit of human replication protein A (hRPA70) was investigated using heteronuclear magnetic resonance spectroscopy. A 1H–15N heteronuclear single quantum coherence (HSQC) titration experiment was performed on a 15N-labeled fragment of hRPA70, containing the N-terminal 168 residues (hRPA701–168) and p53TAD. HRPA701–168 residues important for binding were identified and found to be localized to a prominent basic cleft. This binding site overlapped with a previously identified single-stranded DNA-binding site, suggesting that a competitive binding mechanism may regulate the formation of p53TAD–hRPA70 complex. The amide 1H and 15N chemical shifts of an uniformly 15N-labeled sample of p53TAD were also monitored before and after the addition of unlabeled hRPA701–168. In the presence of unlabeled hRPA701–168, resonance lineshapes increased and corresponding intensity reductions were observed for specific p53TAD residues. The largest intensity reductions were observed for p53TAD residues 42–56. Minimal binding was observed between p53TAD and a mutant form of hRPA701–168, where the basic cleft residue R41 was changed to a glutamic acid (R41E), demonstrating that ionic interactions play an important role in specifying the binding interface. The region of p53TAD most affected by binding hRPA701–168 was found to have some residual alpha helical and beta strand structure; however, this structure was not stabilized by binding hRPA701–168. 15N relaxation experiments were performed to monitor changes in backbone dynamics of p53TAD when bound to hRPA701–168. Large changes in both the transverse (R2) and rotating frame (R1ρ) relaxation rates were observed for a subset of the p53TAD residues that had 1H–15N HSQC resonance intensity reductions during the complex formation. The folding of p53TAD upon complex formation is suggested by the pattern of changes observed for both R2 and R1ρ. A model that couples the formation of a weak encounter complex between p53TAD and hRPA701–168 to the folding of p53TAD is discussed in the context of a functional role for the p53–hRPA70 complex in DNA repair.

Solution structure of the C-terminal X domain of the measles virus phosphoprotein and interaction with the intrinsically disordered C-terminal domain of the nucleoprotein

In this report, the solution structure of the nucleocapsid‐binding domain of the measles virus phosphoprotein (XD, aa 459–507) is described. A dynamic description of the interaction between XD and the disordered C‐terminal domain of the nucleocapsid protein, (NTAIL, aa 401–525), is also presented. XD is an all α protein consisting of a three‐helix bundle with an up‐down‐up arrangement of the helices. The solution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489–491, which is involved in the embedding of the α‐helical XD‐binding region of NTAIL. Secondary chemical shift values for full‐length NTAIL were used to define the precise boundaries of a transient helical segment that coincides with the XD‐binding domain, thus shedding light on the pre‐recognition state of NTAIL. Titration experiments with unlabeled XD showed that the transient α‐helical conformation of NTAIL is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483–506 of NTAIL are in intermediate exchange with XD, while the 475–482 and 507–525 regions are in fast exchange. The NTAIL resonance behavior in the titration experiments is consistent with a complex binding model with more than two states. Copyright

Dynamic behavior of an intrinsically unstructured linker domain is conserved in the face of negligible amino acid sequence conservation.

Proteins or regions of proteins that do not form compact globular structures are classified as intrinsically unstructured proteins (IUPs). IUPs are common in nature and have essential molecular functions, but even a limited understanding of the evolution of their dynamic behavior is lacking. The primary objective of this work was to test the evolutionary conservation of dynamic behavior for a particular class of IUPs that form intrinsically unstructured linker domains (IULD) that tether flanking folded domains. This objective was accomplished by measuring the backbone flexibility of several IULD homologues using nuclear magnetic resonance (NMR) spectroscopy. The backbone flexibility of five IULDs, representing three kingdoms, was measured and analyzed. Two IULDs from animals, one IULD from fungi, and two IULDs from plants showed similar levels of backbone flexibility that were consistent with the absence of a compact globular structure. In contrast, the amino acid sequences of the IULDs from these three taxa showed no significant similarity. To investigate how the dynamic behavior of the IULDs could be conserved in the absence of detectable sequence conservation, evolutionary rate studies were performed on a set of nine mammalian IULDs. The results of this analysis showed that many sites in the IULD are evolving neutrally, suggesting that dynamic behavior can be maintained in the absence of natural selection. This work represents the first experimental test of the evolutionary conservation of dynamic behavior and demonstrates that amino acid sequence conservation is not required for the conservation of dynamic behavior and presumably molecular function.

The structure of the Dead ringer–DNA complex reveals how AT‐rich interaction domains (ARIDs) recognize DNA

The AT‐rich interaction domain (ARID) is a DNA‐binding module found in many eukaryotic transcription factors. Using NMR spectroscopy, we have determined the first ever three‐dimensional structure of an ARID–DNA complex (mol. wt 25.7 kDa) formed by Dead ringer from Drosophila melanogaster. ARIDs recognize DNA through a novel mechanism involving major groove immobilization of a large loop that connects the helices of a non‐canonical helix–turn–helix motif, and through a concomitant structural rearrangement that produces stabilizing contacts from a β‐hairpin. Dead ringers preference for AT‐rich DNA originates from three positions within the ARID fold that form energetically significant contacts to an adenine–thymine base step. Amino acids that dictate binding specificity are not highly conserved, suggesting that ARIDs will bind to a range of nucleotide sequences. Extended ARIDs, found in several sequence‐specific transcription factors, are distinguished by the presence of a C‐terminal helix that may increase their intrinsic affinity for DNA. The prevalence of serine amino acids at all specificity determining positions suggests that ARIDs within SWI/SNF‐related complexes will interact with DNA non‐sequence specifically.

Modeling the accessible conformations of the intrinsically unstructured transactivation domain of p53.

Internuclear distances derived from paramagnetic relaxation enhancement (PRE) data were used to restrain molecular dynamics simulations of the intrinsically unstructured transactivation domain of the tumor suppressor protein, p53. About 1000 structures were simulated using ensemble averaging of replicate molecules to compensate for the inherent bias in the PRE‐derived distances. Gyration radii measurements on these structures show that the p53 transactivation domain (p53TAD) is statistically predominantly in a partially collapsed state that is unlike the open structure that is found for p53TAD bound to either the E3 ubiquitin ligase, MDM2, or the 70 kDa subunit of replication protein A, RPA70. Contact regions that potentially mediate the collapse were identified and found to consist of mostly hydrophobic residues. The identified contact regions preferentially place the MDM2 and RPA70 binding regions in close proximity. We show that our simulations thoroughly sample the available range of conformations and that a fraction of the molecules are in an open state that would be competent for binding either MDM2 or RPA70. We also show that the Stokes radius estimated from the average gyration radius of the ensemble is in good agreement with the value determined using size exclusion chromatography. Finally, the presence of a persistent loop localized to a PXP motif was identified. Serine residues flanking the PXP motif become phosphorylated in response to DNA damage, and we postulate that this will perturb the equilibrium population to more open conformations. Proteins 2008.

Identifying long-range structure in the intrinsically unstructured transactivation domain of p53

Paramagnetic relaxation enhancement (PRE) was used to identify a compact dynamic structure for the intrinsically unstructured transactivation domain of the tumor suppressor protein, p53. Our results show that p53 residues essential for binding to the ubiquitin ligase, MDM2, and the 70 kDa subunit of replication protein A, RPA70, are separated by an average distance of 10–15 Å. This result suggests that a more extended member of the ensemble must be populated prior to binding either MDM2 or RPA70. We also show that PRE can be used to detect intermolecular distances between p53 and RPA70. Proteins 2007.