Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 72-81).

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 113-137).

Guanine nucleotide exchange factors (GEFs) regulate and organize diverse cellular processes through their role in converting GTPases from the inactive GDP bound state to the active GTP bound state. An increasing number of GEFs undergo autoregulatory mechanisms through complex intramolecular interactions. Relief of autoinhibition involves specific phosphorylation or binding to lipid and/or effector proteins at sites distal from the catalytic domain, and is often coupled to membrane recruitment. In Cytohesin Arf GEFs, the catalytic Sec7 domain is autoinhibited by a linker region and C-terminal helix flanking a Pleckstrin Homology (PH) domain. Upon binding of the PH domain to low abundance phosphoinositides, the GTPase Arf6-GTP can both relieve autoinhibition and recruit Cytohesins to the plasma membrane. This thesis focuses on determining the molecular mechanism underlying both these functions. The structural mechanisms by which Arf6-GTP binding relieves autoinhibition were studied using biochemical and crystallographic studies. The crystal structure of the Grp1 PH domain in complex with Arf6 revealed that Arf6-GTP binding relieves autoinhibition through competitive sequestration of the inhibitory elements into grooves formed at the periphery of the interface. Importantly, the interaction orients all known membrane targeting components to a common surface. Detailed biochemical studies showed a common mode of binding among Cytohesin family members in which phosphoinositide head group binding primes the interaction with Arf6, and membrane recruitment of both stimulatory and substrate Arf enhances the effect. To assess changes in the Sec7 domain conformation upon activation, Size Exclusion Chromatography in line with Small Angle X-Ray Scattering (SEC-SAXS) was performed. The unique nature of this data led to the development of a novel data analysis and processing strategy. A graphically based, python-extensible software package was created for data normalization, buffer correction, Guinier Analysis, and constant background subtraction. As an unbiased substitute for traditional buffer subtraction, a method to reconstruct the protein scattering through singular value decomposition (SVD) and linear combination of the basis vectors was developed. These methods produced exceptional data quality and allowed versatility for application to other data collection techniques or systems, especially those lacking confident buffer matching or low signal. SEC-SAXS confirmed the overall structure of autoinhibited Grp1 in solution and showed only slight overall changes upon activation by deletion of the autoinhibitory Cterminal helix. Fusion of Arf6 with Grp1 produced a consistently elongated shape in the active state that was incompatible with the autoinhibited or theoretical active positions of the Sec7 domain. Monte Carlo and rigid body modeling using known structural domains revealed a requirement for Sec7-PH linker flexibility in addition to Sec7 domain mobility. These data support an integrated structural model whereby phosphoinositides and Arf-GTP support nucleotide exchange at membranes through allosteric activation, membrane recruitment, and large-scale rearrangement of the Sec7 domain. Overall, these findings offer insight into Cytohesin function that can be applied to assess relief of autoinhibition in the context of other GEFs and GTPases.

La Proteína Quinasa C (PKC) juega un papel fundamental en la regulación del crecimiento celular. Estas proteínas están implicadas en diferentes vías intracelulares que son consideradas como dianas para el tratamiento contra el cáncer. Atendiendo a las propiedades enzimáticas, las PKC se clasifican en tres grandes subfamilias: clásicas, nuevas y atípicas. En las PKC clásicas, el dominio C2 es un motivo regulador que responde a señales de Ca2+ intracelulares. Este dominio presenta un motivo denominado región rica en lisinas que interacciona con fosfolípidos acídicos. Los resultados de esta tesis demuestran que la afinidad de este dominio por fosfolípidos como el PIP2 es mayor frente a otros de la misma naturaleza. El dominio C2 de las PKC nuevas se une a fosfolípidos cargados negativamente de modo Ca2+-independiente. Nuestro estudio demuestra que la interacción de este dominio con las membranas es principalmente electrostática con una pequeña contribución de interacciones hidrofóbicas. Por otra parte, el estudio de la estructura secundaria del dominio catalítico de la PKC mostró una elevada proporción de hélice . La adición de Mg2+-ATP provocó un mayor efecto protector frente a la desnaturalización térmica. / Protein kinase C (PKC) is a family of related protein kinases that plays an important role in regulating cell growth. These protein kinases are involved in several intracellular pathways that end in transcription and are considered to be potential targets for anticancer therapy. The mammalian isoenzymes have been grouped into three subfamilies according to their enzymatic properties: classical, novel and atypical.The C2 domain is a regulatory sequence motif and is a targeting domain that responds to intracellular Ca2+ signals in classical protein kinases. This domain presents a motif named the lysine-rich cluster that interacts with acidic phospholipids. The results demonstrate that PIP2 interacts with the C2 domain of PKCα in a different way to that described for other phospolipids.C2 domain in novel PKC binds to negatively charged phospholipid vesicles in a Ca2+-independent manner. Our study confirms that the main way in which C2-PKC interacts with membranes is electrostatic in nature, with a very small contribution on the part of hydrophobic interactions.The secondary structure of catalytic domain from atypical PKC showed a high contribution of -helix component. In addition, Mg2+-ATP significantly altered the denaturation pattern of this domain because it protected against denaturation.

HIV/AIDS is a disease of grave global importance with over 33 million people infected world-wide and nearly 2 million deaths each year. The rapid emergence of drug resistance, due to viral mutation, renders anti-retroviral drug candidates ineffective with alarming speed and regularity. Instead of targeting mutation prone viral proteins, an alternative approach is to target host proteins that interact with viral proteins and are critical for the HIV life-cycle. APOBEC3G is a host anti-HIV restriction factor that can exert tremendous negative pressure by hypermutating the viral genome and has the potential to be a promising candidate for anti-retroviral therapeutic research. The work presented in this thesis is focused on investigating the A3G catalytic domain structure and implications of various observed structural features for biological function. High-resolution crystal structures of the A3G catalytic domain were solved using data from macromolecular X-ray crystallographic experiments, revealing a novel intermolecular zinc coordinating motif unique to A3G. Major intermolecular interfaces observed in the crystal structure were investigated for relevance to biochemical activity and biological function. Co-crystallization with a small-molecule A3G inhibitor, discovered using high-throughput screening assays, revealed a cysteine residue near the active site that is critical for inhibition of catalytic activity by catechol moieties. The serendipitous discovery of covalent interactions between this inhibitor and a surface cysteine residue led to further biochemical experiments that revealed the other cysteine, near the active site, to be critical for inhibition. Computational modeling was used to propose a steric-hinderance based mechanism of action that was supported by mutational experiments. Structures of other human APOBEC3 homologs were modeled using in-silico methods examined for similarities and differences with A3G catalytic domain crystal structures. Comparisons based on these homology models suggest putative structural features that may endow substrate specificity and other characteristics to the APOBEC3 family members.