Krishna Mallela | University of Colorado Denver (original) (raw)

Papers by Krishna Mallela

ACS Nano, May 4, 2022

Many aspects of innate immune responses to SARS viruses remain unclear. Of particular interest is... more Many aspects of innate immune responses to SARS viruses remain unclear. Of particular interest is the role of emerging neutralizing antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 in complement activation and opsonization. To overcome challenges with purified virions, here we introduce “pseudovirus-like” nanoparticles with ~70 copies of functional recombinant RBD to map complement responses. Nanoparticles fix complement in RBD-dependent manner in sera of all vaccinated, convalescent and naïve donors, but vaccinated and convalescent donors with the highest levels of anti-RBD antibodies show significantly higher IgG binding and higher deposition of the third complement protein (C3). The opsonization via anti-RBD antibodies is not an efficient process: on average, each bound antibody promotes binding of less than one C3 molecule. C3 deposition is exclusively through the alternative pathway. C3 molecules bind to protein deposits, but not IgG, on the nanoparticle surface. Lastly, “pseudovirus-like” nanoparticles promote complement-dependent uptake by granulocytes and monocytes in the blood of vaccinated donors with high anti-RBD titers. Using nanoparticles displaying SARS-CoV-2 proteins, we demonstrate subject-dependent differences in complement opsonization and immune recognition.

bioRxiv (Cold Spring Harbor Laboratory), Jun 14, 2023

With hundreds of coronaviruses (CoVs) identified in bats that are capable of infecting humans, it... more With hundreds of coronaviruses (CoVs) identified in bats that are capable of infecting humans, it is important to understand how CoVs that affected the human population have evolved. Seven known coronaviruses have infected humans, of which three CoVs caused severe disease with high mortality rates: SARS-CoV emerged in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Both SARS-CoV and SARS-CoV-2 belong to the same family, follow the same receptor pathway, and use their receptor binding domain (RBD) of spike protein to bind to the ACE2 receptor on the human epithelial cell surface. The sequence of the two RBDs is divergent, especially in the receptor binding motif (RBM) that directly interacts with ACE2. We probed the biophysical differences between the two RBDs in terms of their structure, stability, aggregation, and function. Since RBD is being explored as an antigen in protein subunit vaccines against CoVs, determining these biophysical properties will also aid in developing stable protein subunit vaccines. Our results show that despite RBDs having a similar three-dimensional structure, they differ in their thermodynamic stability. RBD of SARS-CoV-2 is significantly less stable than that of SARS-CoV. Correspondingly, SARS-CoV-2 RBD shows a higher aggregation propensity. Regarding binding to ACE2, less stable SARS-CoV-2 RBD binds with a higher affinity than more stable SARS-CoV RBD. In addition, SARS-CoV-2 RBD is more homogenous in terms of its binding stoichiometry towards ACE2, compared to SARS-CoV RBD. These results indicate that SARS-CoV-2 RBD differs from SARS-CoV RBD in terms of its stability, aggregation, and function, possibly originating from the diverse RBMs. Higher aggregation propensity and decreased stability of SARS-CoV-2 RBD warrants further optimization of protein subunit vaccines that use RBD as an antigen either by inserting stabilizing mutations or formulation screening. Statement of Significance This study holds significant relevance in the context of the COVID-19 pandemic and the broader understanding of coronaviruses. A comparison of the receptor binding domains (RBDs) of SARS-CoV and SARS-CoV-2 reveals significant differences in their structure, stability, aggregation, and function. Despite divergent sequences, the RBDs share a similar fold and ACE2 receptor binding capability, likely through convergent evolution. These findings are crucial for understanding coronavirus evolution, interactions with human receptors, and the spillover of coronaviruses from animals to humans. The study also has implications for vaccine design strategies for SARS-CoVs, where the RBD is used as an antigen in protein subunit vaccines. By anticipating future outbreaks and enhancing our understanding of zoonotic spillover, this research contributes to safeguarding human health. .

Circulation Research, Aug 1, 2013

Muscular dystrophy (MD) is an incurable disease, and affects all types of muscles. The decreased ... more Muscular dystrophy (MD) is an incurable disease, and affects all types of muscles. The decreased function of heart muscles causes heart problems such as cardiomyopathy and congestive heart failure. A deficiency in functional dystrophin protein in muscle cells triggers the onset of Duchenne MD and Becker MD. Utrophin is the closest homologue of dystrophin and is being tested as a protein drug to replace the loss of functional dystrophin in human patients. However, utrophin is less stable and binds to actin through a different mode of contact when compared with dystrophin. To optimize utrophin as the protein drug, we first need to understand how its molecular structure controls its stability and function. Utrophin and dystrophin bind to actin using tandem calponin-homology (CH) domains at their N-terminus. Individual CH domains of utrophin and dystrophin are very similar in amino acid sequence and their three dimensional structures. The major difference is in their relative orientation around the linker region that connects the two CH domains. To determine the role of the linker, we designed constructs with linkers switched between utrophin and dystrophin. Our results indicate that the tandem CH domain of utrophin with dystrophin linker (UDL) is more stable than that of utrophin but less stable than that of dystrophin. Similarly, tandem CH domain of dystrophin with utrophin linker (DUL) is less stable than that of dystrophin but more stable than that of utrophin. Kinetic folding and unfolding studies suggest that the linker predominantly affects the folding rate rather than the unfolding rate. In addition to stability and folding, the linker also controls protein aggregation. UDL is more prone to aggregation when compared with that of utrophin, whereas DUL is less prone to aggregation when compared with that of dystrophin. These results indicate that the linker plays a major role in controlling the stability and function of tandem CH domains, and further suggests that it is possible to engineer utrophin to behave precisely like dystrophin based on their molecular structures.

Biophysical Journal, 2010

proton channels from influenza A and B viruses by solution NMR spectroscopy. The channel structur... more proton channels from influenza A and B viruses by solution NMR spectroscopy. The channel structures reveal pore features that are important for proton gating and proton relay. Structural details of the anti-influenza drug, rimantadine, bound to the channel suggests an unexpected allosteric mechanism of drug inhibition and drug resistance, which has been verified by thorough functional and mutagenesis experiments. The work is supported by NIH grant AI067438.

ACS omega, Jan 10, 2020

Genetic mutations in Duchenne muscular dystrophy (DMD) gene affecting the expression of dystrophi... more Genetic mutations in Duchenne muscular dystrophy (DMD) gene affecting the expression of dystrophin protein lead to a number of muscle disorders collectively called dystrophinopathies. In addition to muscle dystrophin, mutations in brain-specific dystrophin isoforms, in particular those that are expressed in the brain cortex and Purkinje neurons, result in cognitive impairment associated with DMD. These isoforms carry minor variations in the flanking region of the N-terminal actin-binding domain (ABD1) of dystrophin, which is composed of two calponin-homology (CH) domains in tandem. Determining the effect of these sequence variations is critical for understanding the mechanisms that govern varied symptoms of the disease. We studied the impact of differences in the N-terminal flanking region on the structure and function of dystrophin tandem CH domain isoforms. The amino acid changes did not affect the global structure of the protein but drastically affected the thermodynamic stability, with the muscle isoform more stable than the brain and Purkinje isoforms. Actin binding investigated with actin from different sources (skeletal muscle, smooth muscle, cardiac muscle, and platelets) revealed that the muscle isoform binds to filamentous actin (F-actin) with a lower affinity compared to the brain and Purkinje isoforms, and a similar trend was observed with actin from different sources. In addition, all isoforms showed a higher affinity to smooth muscle actin in comparison to actin from other sources. In conclusion, tandem CH domain isoforms might be using minor sequence variations in the N-terminal flanking regions to modulate their thermodynamic stability and actin-binding function, thus leading to specificity in dystrophin−actin interactions in various tissues.

Biophysical Journal, 2012

twisting of the filaments. In many mechanical cellular activities, such as cell movements, divisi... more twisting of the filaments. In many mechanical cellular activities, such as cell movements, division, and controls of cell shape, modulation of filament stretching and twisting dynamics has been linked to regulatory actin-binding protein function. For example, mechanical stretching under tension causes structural changes in twist because of the mechanical characteristic of double helical filaments, that might prevent cofilin from binding to the actin filament. Therefore, it is important to quantitatively evaluate the stretching-twisting coupling behaviors of actin filaments for better understanding of actin dynamics. This study investigates the stretching-twisting coupling behaviors using molecular dynamics simulations. A model for the actin filament consisting of fourteen actin subunits in an ionic solvent was constructed as a minimal functional unit. To evaluate the stretching-twisting coupling behaviors, longitudinal and twisting Brownian motions of the filament were analyzed. The result demonstrated that the longitudinal and twisting motions of the filament exhibit a strong correlation, which indicate that the double-helix structure was untwisted under tensile force. The results obtained from this study contribute to the understanding of mechanochemical interactions concerning actin dynamics, showing that increased tensile force in the filament prevents actin regulatory proteins from binding to the filament.

Bioscience Reports, Nov 1, 2022

Alzheimer's disease (AD) is one of the most prominent neurodegenerative diseases. Results from an... more Alzheimer's disease (AD) is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NAD(P)H quinone oxidoreductase 1 (NQO1) may accelerate the aggregation of Alzheimer's amyloid-β peptide (Aβ 1-42). Here, we examined in vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Aβ 1-42 and modify its rate of aggregation. When monitored using the fluorescence of either noncovalent thioflavin T (ThT) or HiLyte Fluor 647 (HF647) dye covalently attached to the Aβ 1-42 peptide, the aggregation kinetics of Aβ 1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type (WT) NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor that binds near the FAD-binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Aβ 1-42. Imaging flow cytometry confirmed in-vitro coaggregation of NQO1 isoforms and Aβ 1-42. Aβ 1-42 alone forms rod-shaped fibril structures while in the presence of NQO1 isoforms, Aβ 1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry (ITC) analysis indicates that Aβ 1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of coaggregates of Aβ 1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.

Journal of Molecular Biology, Jul 1, 2022

Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe... more Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe symptoms and increased mortality among infected people. Our study seeks to examine how the biophysical parameters of the Delta variant correlate to the clinical observations. Receptor binding domain (RBD) is the first point of contact with the human host cells and is the immunodominant form of the spike protein. Delta variant RBD contains two novel mutations L452R and T478K. We examined the effect of single as well as the double mutations on RBD expression in human Expi293 cells, RBD stability using urea and thermal denaturation, and RBD binding to angiotensin converting enzyme 2 (ACE2) receptor and to neutralizing antibodies using isothermal titration calorimetry. Delta variant RBD showed significantly higher expression compared to the wild-type RBD, and the increased expression is due to L452R mutation. Despite their non-conservative nature, none of the mutations significantly affected RBD structure and stability. All mutants showed similar binding affinity to ACE2 and to Class 1 antibodies (CC12.1 and LY-CoV016) as that of the wild-type. Delta double mutant L452R/T478K showed no binding to Class 2 antibodies (P2B-2F6 and LY-CoV555) and a hundred-fold weaker binding to a Class 3 antibody (REGN10987), and the decreased antibody binding is determined by the L452R mutation. These results indicate that the immune escape from neutralizing antibodies, rather than increased receptor binding, is the main biophysical parameter that determined the fitness landscape of the Delta variant RBD.

Molecular Pharmaceutics, Sep 4, 2019

Two of the most common forms of chemical modifications that may compromise the efficacy of therap... more Two of the most common forms of chemical modifications that may compromise the efficacy of therapeutic proteins are the deamidation of asparagine residues and oxidation of methionine residues. We probed how deamidation affects the structure, stability, aggregation, and function of interferon alpha-2a (IFNA2a), and compared with our earlier results on methionine oxidation. Upon deamidation, no significant changes were observed in the global secondary structure of IFNA2a with minor changes in its tertiary structure. However, deamidation destabilized the protein, and increased its propensity to aggregate under accelerated stress conditions. Cytopathic inhibition and anti-proliferation assays showed drastic decrease in the functionality of deamidated IFNA2a compared to the wild-type. 2D NMR measurements showed structural changes in local protein regions, with no effect on the overall global structure of IFNA2a. These local protein regions corresponded well with the aggregation hot-spots predicted by computational programs, and the functional hot-spots identified by site-directed mutagenesis. When compared to the effects of methionine oxidation, deamidation caused lesser aggregation, because of lesser structural changes observed in aggregation hot-spots by 2D NMR. In comparison to oxidation, deamidation showed larger decrease in function, because deamidation affected key amino acid residues in functional hot-spots as observed by 2D NMR and structural modeling. Such quantitative comparison between the effects of deamidation and oxidation on a pharmaceutical protein has not been done before, and the high-resolution structural information of local protein regions obtained by 2D NMR provided a better insight compared to low-resolution methods that probe global protein structure.

Journal of Pharmaceutical Sciences, Nov 1, 2018

Current guidelines indicate that the effects of oxidation should be included as part of forced de... more Current guidelines indicate that the effects of oxidation should be included as part of forced degradation studies on protein drugs. We probed the effect of 3 commonly used oxidants, hydrogen peroxide, tert-butyl hydroperoxide, and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), on a therapeutic monoclonal IgG1 antibody (mAb8). Upon oxidation, mAb8 did not show noticeable changes in its secondary structure but showed minor changes in tertiary structure. Significant decrease in conformational stability was observed for all the 3 oxidized forms. Both hydrogen peroxide and tert-butyl hydroperoxide destabilized mainly the C H 2 domain, whereas AAPH destabilized the variable domain in addition to C H 2. Increased aggregation was found for AAPH-oxidized mAb8. In addition, a significant decrease in Fc receptor binding was observed for all 3 oxidized forms. Antibody dependent cell-mediated cytotoxicity, binding to target protein receptor, and cell proliferation activity were significantly reduced in the case of AAPH-oxidized mAb8. The presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation. The results of this study show that the 3 oxidants differ in terms of their effects on the structure and function of mAb8 because of chemical modification of different sets of residues located in Fab versus Fc.

International Journal of Pharmaceutics, Aug 1, 2018

Photostability testing of therapeutic proteins is a critical requirement in the development of bi... more Photostability testing of therapeutic proteins is a critical requirement in the development of biologics. Upon exposure to light, pharmaceutical proteins may undergo a change in structure, stability, and functional properties that could have a potential impact on safety and efficacy. In this work, we studied how exposure to light, according to ICH guidelines, leads to photo-oxidation of a therapeutic IgG1 mAb. We also tested the ability of five different excipients to prevent such oxidation. In samples that were exposed to light, we found that the C2 domain was considerably destabilized but there were no major changes in the overall structure of the protein. Aggregation of the protein was observed because of light exposure. Mass spectrometry identified that light exposure oxidizes two key methionine residues in the Fc region of the protein. In terms of function, a loss in binding to the neonatal Fc receptor, decreased antibody-dependent cell-mediated cytotoxicity and cell proliferation activities of the protein were seen. Combined analysis of the photo-oxidation effects on the structure, stability, aggregation, and function of the mAb has identified the underlying unifying mechanism. Among the sugars and amino acids tested, methionine was the most effective in protecting mAb against photo-oxidation.

Circulation Research, Jul 18, 2014

Tandem calponin-homology (CH) domains constitute a major class of actin-binding domains that incl... more Tandem calponin-homology (CH) domains constitute a major class of actin-binding domains that include dystrophin and utrophin, the two key proteins involved in muscular dystrophy. Despite their importance, how their structure controls their function is not understood. Here, we study the contribution of individual CH domains to the actin-binding function and thermodynamic stability of utrophin’s tandem CH domain. Traditional actin co-sedimentation assays indicate that the isolated C-terminal CH2 domain binds weakly to F-actin when compared with the full-length tandem CH domain. In contrast, isolated CH1 binds to F-actin with a similar efficiency as that of the full-length tandem CH domain. Thus, the obvious question that arises is why tandem CH domains require CH2, when their actin-binding efficiency is originating primarily from CH1. To answer, we probed the thermodynamic stabilities of individual CH domains. Isolated CH1 domain is unstable and is prone to serious aggregation. Isolated CH2 is very stable, even appears to be more stable than the full-length tandem CH domain. In addition, the CH2 domain, which is more stable, is less functional. These results indicate that the main function of CH2 is to stabilize CH1. Consistently, the proposed structure of utrophin’s tandem CH domain based on earlier X-ray studies indicates a close proximity between the C-terminal helix of CH2 and the N-terminal helix of CH1, and this helix in CH2 is more dynamic in the full-length protein when compared with that in the absence of CH1, suggesting the mechanism by which CH2 stabilizes CH1. These observations indicate that the two CH domains contribute differentially to the folding and function of tandem CH domains, although both domains essentially have the same native structure in the tandem CH domain. The N-terminal domain determines the function, whereas the C-terminal domain determines the stability. This work was funded by the AHA Grant 11SDG4880046.

Biophysical Journal, 2015

Protein folding, the self-assembly of a protein molecule or domain into a tertiary structure, can... more Protein folding, the self-assembly of a protein molecule or domain into a tertiary structure, can occur as a protein molecule is being synthesized by the ribosome in a process referred to as co-translational folding. The most convincing demonstration that co-translational folding occurs inside cells comes from pulse-chase experiments in which the synthesis of the cytosolic Semliki Forest virus protein (SFVP) was monitored in Chinese hamster ovarian cells [1].

Journal of Pharmaceutical Sciences, Feb 1, 2015

Benzyl alcohol (BA) is the most widely used antimicrobial preservative in multidose protein formu... more Benzyl alcohol (BA) is the most widely used antimicrobial preservative in multidose protein formulations, and has been shown to cause protein aggregation. Our previous work on a model protein cytochrome c demonstrated that this phenomenon occurs via partial unfolding. Here, we examine the validity of these results by investigating the effect of BA on a pharmaceutically relevant protein, interferon ␣-2a (IFNA2). IFNA2 therapeutic formulations available on the pharmaceutical market contain BA as a preservative. Isothermal aggregation kinetics and temperature scanning demonstrated that BA induced IFNA2 aggregation in a concentration-dependent manner. With increasing concentration of BA, the apparent aggregation temperature of IFNA2 linearly decreased. Denaturant melts measured using protein intrinsic fluorescence and that of the 1-anilinonaphthalene-8-sulfonic acid dye indicated that IFNA2 stability decreased with increasing BA concentration, populating a partially unfolded intermediate. Changes in nuclear magnetic resonance chemical shifts and hydrogen exchange rates identified the structural nature of this intermediate, which correlated with an aggregation "hot-spot" predicted by computational methods. These results indicate that BA induces IFNA2 aggregation by partial unfolding rather than global unfolding of the entire protein, and is consistent with our earlier conclusions from model protein studies.

International Journal of Pharmaceutics, Sep 1, 2014

Antimicrobial preservatives (APs) are included in liquid multi-dose protein formulations to comba... more Antimicrobial preservatives (APs) are included in liquid multi-dose protein formulations to combat the growth of microbes and bacteria. These compounds have been shown to cause protein aggregation, which leads to serious immunogenic and toxic side-effects in patients. Our earlier work on a model protein cytochrome c (Cyt c) demonstrated that APs cause protein aggregation in a specific manner. The aim of this study is to validate the conclusions obtained from our model protein studies on a pharmaceutical protein. Interferon α-2a (IFNA2) is available as a therapeutic treatment for numerous immune-compromised disorders including leukemia and hepatitis c, and APs have been used in its multi-dose formulation. Similar to Cyt c, APs induced IFNA2 aggregation, demonstrated by the loss of soluble monomer and increase in solution turbidity. The extent of IFNA2 aggregation increased with the increase in AP concentration. IFNA2 aggregation also depended on the nature of AP, and followed the order m-cresol > phenol > benzyl alcohol > phenoxyethanol. This specific order exactly matched with that observed for the model protein Cyt c. These and previously published results on antibodies and other recombinant proteins suggest that the general mechanism by which APs induce protein aggregation may be independent of the protein.

Journal of Pharmaceutical Sciences, Jul 1, 2021

Adeno-associated virus (AAV) has emerged as a leading platform for gene delivery for treating var... more Adeno-associated virus (AAV) has emerged as a leading platform for gene delivery for treating various diseases due to its excellent safety profile and efficient transduction to various target tissues. However, the large-scale production and long-term storage of viral vectors is not efficient resulting in lower yields, moderate purity, and shorter shelf-life compared to recombinant protein therapeutics. This review provides a comprehensive analysis of upstream, downstream and formulation unit operation challenges encountered during AAV vector manufacturing, and discusses how desired product quality attributes can be maintained throughout product shelf-life by understanding the degradation mechanisms and formulation strategies. The mechanisms of various physical and chemical instabilities that the viral vector may encounter during its production and shelf-life because of various stressed conditions such as thermal, shear, freeze-thaw, and light exposure are highlighted. The role of buffer, pH, excipients, and impurities on the stability of viral vectors are also discussed. As such, the aim of this review is to outline the tools and a potential roadmap for improving the quality of AAV-based drug products by stressing the need for a mechanistic understanding of the involved processes.

Journal of Pharmaceutical Sciences, Feb 1, 2013

One-third of protein formulations are multi-dose. These require antimicrobial preservatives (APs)... more One-third of protein formulations are multi-dose. These require antimicrobial preservatives (APs); however, some APs have been shown to cause protein aggregation. Our previous work on a model protein cytochrome c indicated that partial protein unfolding, rather than complete unfolding, triggers aggregation. Here, we examined the relative strength of five commonly used APs on such unfolding and aggregation, and explored whether stabilizing the aggregation "hot-spot" reduces such aggregation. All APs induced protein aggregation in the order m-cresol > phenol > benzyl alcohol > phenoxyethanol > chlorobutanol. All these enhanced the partial protein unfolding that includes a local region which was predicted to be the aggregation "hot-spot". The extent of destabilization correlated with the extent of aggregation. Further, we show that stabilizing the "hot-spot" reduces aggregation induced by all five APs. These results indicate that m-cresol causes the most protein aggregation, whereas chlorobutanol causes the least protein aggregation. The same protein region acts as the "hot-spot" for aggregation induced by different APs, implying that developing strategies to prevent protein aggregation induced by one AP will also work for others.

bioRxiv (Cold Spring Harbor Laboratory), May 24, 2021

Emergence of new SARS-CoV-2 variants has raised concerns at the effectiveness of vaccines and ant... more Emergence of new SARS-CoV-2 variants has raised concerns at the effectiveness of vaccines and antibody therapeutics developed against the unmutated wild-type virus. We examined the effect of 12 most commonly occurring mutations in the receptor binding domain on its expression, stability, activity, and antibody escape potential-some of the factors that may influence the natural selection of mutants. Recombinant proteins were expressed in human cells. Stability was measured using thermal denaturation melts. Activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to ACE2 and to a neutralizing human antibody CC12.1, respectively. Our results show that variants differ in their expression levels with the two least stable variants showing lesser expression. Out of the 8 wellexpressed mutants, only 2 (N501Y and K417T/E484K/N501Y) showed stronger affinity to ACE2, 4 (Y453F, S477N, T478I and S494P) have similar affinity, whereas the other 2 (K417N and E484K) have weaker affinity when compared to the wild-type. In terms of CC12.1 binding, when compared to the wild-type, 4 variants (K417N, Y453F, N501Y and K417T/E484K/N501Y) have weaker affinity, 2 (S477N and S494P) have similar affinity, and 2 (T478I and E484K) have stronger affinity. Taken together, these results indicate that multiple factors contribute towards the natural selection of variants, and all these factors need be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant. .

Biophysical Journal, 2014

The use of modular protein domains has emerged as a prominent feature of increasing phylogenetic ... more The use of modular protein domains has emerged as a prominent feature of increasing phylogenetic complexity. Linking modular domains within a single protein allows complex regulation while conserving the sequence and structure of the individual domains. For instance, spatio-temporal control of signaling proteins is often achieved by stringing together a conserved catalytic domain with one or more regulatory modules. These modules can play multiple roles including masking the catalytic site to inhibit basal activity (auto-inhibition), releasing auto-inhibition through conformational changes triggered by second messenger stimuli, and facilitating translocation to subcellular compartments through binding secondary messengers or scaffolding proteins. Each additional module in a signaling protein provides a combinatorial enhancement to its regulation and cellular function. The protein-context independent structure and cellular function of individual modules have been extensively researched using biophysical approaches such as x-ray crystallography and NMR. Most modular domains have an evolutionarily conserved canonical function. However, coordination of interactions between these domains remains largely unexplored primarily due to the reliance on reductionist structural and biochemical approaches. As a corollary, our current structural understanding of modular signaling proteins does not adequately address the versatility of their cellular function. Using the uniquely persistent ER/K a-helix derived from the lever arm of myosin VI combined with genetically encoded fluorophores we have previously developed a methodology termed SPASM to both observe and modulate intra-molecular interactions between domains in multi-domain proteins. Using human protein kinase C a (PKCa) as a model multi-domain signaling protein, we have uncovered intra-and inter-molecular interactions involving each of its modular domains. These interactions contribute to context-dependent spatio-temporal regulation of PKC function in cells. Our findings highlight the importance of intra-molecular interactions in biologically critical multi-domain proteins.