Browsing by Subject "Protein Structure, Tertiary"
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Item Characterization of Internal Dynamics in Vav1: Method Development, Mutual Coupling and Functional Relevance(2009-09-04) Li, Pilong; Rosen, Michael K.Protein motions are important to activity, but quantitative relationships between internal dynamics and function are not well understood. The Dbl homology (DH) domain of the proto-oncoprotein and guanine nucleotide exchange factor Vav1 is autoinhibited through interactions between its catalytic surface and a helix from an N-terminal acidic region. Phosphorylation of the helix relieves autoinhibition. Here I show by NMR spectroscopy that the autoinhibited DH domain (AD) exists in equilibrium between a ground state, where the active site is blocked by the inhibitory helix, and an excited state, where the helix is dissociated. Across a series of mutants that differentially sample these states, catalytic activity of the autoinhibited protein and its rate of phosphorylation are linearly dependent on the population of the excited state. Thus, internal dynamics are required for and control both basal activity and the rate of full activation of the autoinhibited DH domain. Vav1 belong to a class of multi-domain signaling proteins exhibit complex behaviors due to cooperative interactions between domains. In many such proteins there is a core regulatory interaction, involving binding of an inhibitory element to the active site of a functional domain like the inhibitory helix to DH in Vav1. The core interaction is cooperatively enhanced by additional intramolecular domain-domain contacts. The physical basis of this cooperativity, and thus the energetic construction of multi-domain systems, is not well understood. Dynamics analysis of AD reveals that the closed and open populations are about 10:1 for the core interaction in isolation. In the full five-domain regulatory fragment of Vav1, interactions between domains outside of the core further bias this inhibitory equilibrium ~10-fold toward the closed state, further suppressing activity. Thus, Vav1 is controlled by two, weakly biasing, but thermodynamically coupled equilibria--an energetic construction that is probably general among multi-domain proteins. The dynamic landscape of AD is composed of two ?s-ms time scale motions: one is the inhibitory helix binding to and dissociating from the DH domain and another is intrinsic to the DH domain. Interestingly relative populations and exchange rates of the second process are altered upon perturbations to the inhibitory helix, suggesting that the two dynamic processes are energetically and kinetically coupled. A strategy has been established to quantify the thermodynamic and kinetic coupling strengths between the two processes via direction parameterization of four-state equilibria using NMR Carr-Purcell-Meiboom-Gill measurement. The coupling strengths between the two dynamic processes in AD are 1.0~1.5 kcal M-1 comparable to the coupling strength between the modulatory interaction and the helix-DH interactions in the full five-domain regulatory fragment of Vav1. The coupling strength is relatively weak consistent with the coupling strengths reported for many other signaling proteins such as Src tyrosine kinase. These findings suggest that weakly coupling may be a common theme in regulatory molecules.Item Chemical Footprinting of Polymeric Structure of hnRNPA2 Low Complexity Domain(2016-05-24) Xiang, Siheng; Nijhawan, Deepak; Rosen, Michael K.; Yu, Hongtao; Kliewer, Steven A.; McKnight, Steven L.Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low complexity domains. These low complexity sequences have been shown to induce phase transition in low salt buffer. When incubated at high concentration, certain of these low complexity domains polymerize into labile, amyloid-like fibers. I developed a chemical footprinting method to probe solvent accessible residues in the low complexity domain polymers. By acetylating protein side chains with N-acetylimidazole, and comparing the acetylation in native and denatured conformation by use of SILAC mass spectrometry, I generated an NAI footprint for hnRNPA2 polymers. I deployed this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, and offered evidence that its low complexity domain exists in a similar conformation as that described for recombinant polymers of the protein. To study the structure of the low complexity sequence in liquid-like droplets, I systematically mutated individual tyrosine or phenylalanine residues to serine, assayed the ratio of these mutants that partitioned into the droplet phase, and compared the results with their abilities to grow polymeric fibers from wild-type seeds. The same region which contained mutations impeding fiber growth were found to display decreased partitioning into liquid-like droplets. Additionally, the NAI footprint of hnRNPA2 in these liquid-like droplets appeared to be similar to the footprint found in fibers. These observations suggest that the hnRNPA2 low complexity domain adopts a similar structure in amyloid-like fibers and liquid-like droplets. Combining these results, my studies favor the perspective that cross-beta polymerization commonly drives the formation of hydrogels, the retention of low complexity domains trapped by hydrogels, the formation of liquid-like droplets, the partitioning of low complexity domains into existing liquid-like droplets, and the formation and maturation of RNA granules. In other words, my results provide evidence that the involvement of low complexity domains in the formation of RNA granules, liquid-like droplets and hydrogels all rely on one in the same phenomenon - cross-beta polymerization.Item Evolutionary Classification of Protein Domains: From Remote Homology to Family(2017-11-20) Liao, Yuxing; Rizo-Rey, José; Grishin, Nick V.; Rice, Luke M.; Tomchick, Diana R.Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. A protein domain classification splits protein into domains and organizes them according to their evolutionary history. Existing classification databases fall back the speed of protein structure determination and do not include some known homologous relationships. I have participated in creating a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures and developed a website for easy access and searches with keyword, sequence or structure (http://prodata.swmed.edu/ecod). ECOD (Evolutionary Classification Of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or fold). Our database uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary relationships among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. The classification is assisted by an automated pipeline that classifies the most of new structures in Protein Data Bank weekly. This synchronization uniquely distinguishes ECOD among all protein classifications. For proteins that lack confident results from the automatic pipeline, I rely on information from literature, sequence and structure similarity scores, visual comparison and experience to classify them manually. I document the manual curation process in detail with an example of the remote homology between an autoproteolytic domain found in GPCR-Autoproteolysis Inducing domain, ZU5 and nucleoporin98. ECOD also recognizes closer relationships at the family level, initially with Pfam families. However, existing family databases do not cover all structures and disagree with ECOD in terms of domain definition and boundary. I generate multiple sequence alignment and profile for domains in the same family with structural information and demonstrate that the alignment quality is similar to manually checked Pfam seed alignments. I compare ECOD family profiles with Pfam and Conserved Domain Database and discuss about the improvement of domain boundary over known families and the dominance of small families in new families.Item Regulation of Endocytosis of ROMK Channel by WNK Kinase Family(2009-09-04) Wang, Hao-Ran; Huang, Chou-LongWNK kinases are members of a novel family of serine/threonine kinases with atypical placement of the catalytic lysine. Mutations in WNK1 and WNK4 cause pseudohypoaldosteronism type 2 (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. Renal outer medullary potassium channel (ROMK) is responsible for constitutive K+ secretion in the kidney. WNK1 and WNK4 stimulate the clathrin-mediated endocytosis of ROMK, which contributes to the pathogenesis of hyperkalemia in PHAII patients. Intersectin (ITSN) is a multimodular endocytic scaffold protein. The proline-rich domains of WNK1 and WNK4 bind with the Src-homology domain (SH3) of intersectin, and this interaction is important for the stimulation of endocytosis of ROMK by WNKs. Intersectin will further activate the GTPase activity of dynamin and the actin polymerization of N-WASP, and thus promote endocytosis of ROMK channel. WNK1 inhibition of ROMK is further regulated by the kinase domain conformation, which is critical for WNK1 to recruit intersectin. A shorter renal alternatively spliced form of WNK1 that lacks the kinase domain, known as kidney specific WNK1 (KSWNK1),interacts with WNK1 kinase domain and antagonizes WNK1 inhibition of ROMK. The 4a domain and the auto-inhibitory domain in KS-WNK1 are responsible for the antagonization. The antagonism of WNK1 by 4a domain of KS-WNK1 can be abolished by 2-BP (a palmitoylation inhibitor) and hydrogen peroxide (generated during K+ deficiency). These results provide a molecular mechanism for the regulation of endocytosis of ROMK by WNK kinase family.Item Structural Analysis of Domain Swapping in the Protein Kinase: Crystal Structure of Human Ste20 OSR1 Kinase Domain(2007-12-18) Lee, Seung-Jae; Goldsmith, Elizabeth J.Ste20p (Sterile 20 protein) is a yeast MAP4K involved in the pheromoneresponsive MAPK cascade of the mating pathway. Recent studies reveal that its homologs in mammals, Drosophila melanogaster, Caenorhabditis elegans and other organisms constitute a large emerging group of protein kinases including 28 members in human. The Ste20p family has gained remarkable interest, due to the recent finding that it has various intracellular regulatory effects including the regulation of apoptosis and rearrangement of the cytoskeleton triggering cell-shape changes and cell motility. Moreover, from the viewpoint of structural biology, it is intriguing that the Ste20p family is characterized by the presence of a conserved kinase domain and a noncatalytic region of great diversity at various locations. Despite recent structural studies of Ste20p kinases such as PAK1 or TAO2, the mechanism of kinase regualtion in this family still remains to be investigated. Thus, in order to deepen our understanding of Ste20p from a structural viewpoint, the OSR1 kinase, which belongs to the GCK VI subfamily, was crystallized. In this dissertation, I present the crystal structure of the OSR1 kinase domain in an inactive conformation. This crystallographic result demonstrates the unexpected finding that the OSR1 kinase domain is dimerized in domain-swapped manner, which is a novel mode of protein-protein interaction in the protein kinase family. The detailed structural analysis shows that alpha EF helix and the P+1 loop region located in the activation segment were completely swapped between two monomers. The DFG magnesium binding loop and the N-terminal F-helix seem to function as anchors for the hinge loop, based upon the structural alignment with other domain-swapped Ste20p kinases, which were reported after I completed the OSR1 structure determination. Several Ste20p kinases also form domain-swapped dimers in a strikingly similar manner to OSR1.Item Structural and Mechanistic Studies of Two Regulatory Factors in Actin Cytoskeletal Signaling: Vav and VopL(2011-12-15) Yu, Bingke; Rosen, Michael K.Proper control of actin cytoskeletal dynamics is essential for cell survival. The goals of my thesis work have been to characterize the structural and biophysical properties of two regulatory proteins in actin cytoskeletal rearrangement pathway: Vav and Vibrio outer protein L (VopL). Vav proteins are guanine nucleotide exchange factors for Rho family GTPases. They play key roles in actin regulatory pathways and control diverse cellular processes like T cell maturation and activation, cell migration and phagocytosis. They belong to a group of multi-domain signaling proteins which display complex behaviors because of the collective regulation from multiple domains. Previous work has shown that Vav is autoinhibited in the resting state through the cooperative suppression of N-terminal Calponin domain and Acidic region, with the physical mechanism yet to be determined. Here through structural, energetic and biochemical studies, I demonstrate that the Calponin homology domain of Vav binds to the Pleckstrin homology domain, restrains the inhibitory helix in the Acidic region, and shifts the Dbl homology domain - inhibitory helix equilibrium to a more closed state. This construction enables strong suppression and an efficient activation process. The energetic basis of Vav autoinhibition may turn out to be widespread in multi-domain systems. VopL, a pathogenic effector from Vibrio parahaemalyticus, is an actin nucleation factor that induces stress fibers during bacterial infection. It contains three N-terminal Wiskott-Aldrich Homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). It potently promotes actin filament nucleation in vitro. However, the physical basis of VopL mediated nucleation has not been understood. Here I performed structural and biochemical studies to investigate the mechanism of actin filament nucleation by VopL. I found that both the WH2 element and VCD are required for VopL activity. The crystal structure of VCD revealed a U-shaped dimer that is stabilized by a terminal coiled-coil. Dimerization of the WH2 motifs as well as contacts between VCD and actin contribute to the nucleation activity of VopL. My studies suggest the formation of a structurally organized actin cluster involving lateral contacts during nucleation. Stabilization of these lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.Item Studies on Combining Sequence and Structure for Protein Classification(2010-01-12) Kim, Bong-Hyun; Grishin, Nick V.The ultimate goal of our research is to develop a better understanding of how proteins evolve different structures and functions. A large scale protein clustering can provide a useful platform to identify such principles of protein evolution. Manual classification schemes accurately group homologous proteins, but they are slow and subjective. Automatic protein clustering methods are largely based on sequence information. Therefore, they often do not accurately reflect remote homologies that can be recognized by structural information. We hypothesized that combining evolutionary signals from protein sequence and 3D structure will improve automated protein classification. To test this hypothesis, we clustered proteins into evolutionary groups using both sequence and structure by a fully automated method. We developed a stringent algorithm, self-consistency grouping (SCG) method, which clusters proteins if all the proteins in the group are more similar to each other than to proteins outside the group. Comparison of SCG and other commonly used clustering methods to a widely accepted manual classification scheme, Structural Classification of Protein (SCOP), showed SCG groups to better reflect the reference classification. In depth analysis of SCG clusters highlights new non-trivial evolutionary links between proteins. SCG clustering can be further developed as a reference for evolutionary classification of proteins.