Characterizing the Diversity of the CDR-H3 Loop Conformational Ensembles in Relationship to Antibody Binding Properties

We present an approach to assess antibody CDR-H3 loops according to their dynamic properties using molecular dynamics simulations. We selected six antibodies in three pairs differing substantially in their individual promiscuity respectively specificity. For two pairs of antibodies crystal structures are available in different states of maturation and used as starting structures for the analyses. For a third pair we chose two antibody CDR sequences obtained from a synthetic library and predicted the respective structures. For all three pairs of antibodies we performed metadynamics simulations to overcome the limitations in conformational sampling imposed by high energy barriers. Additionally, we used classic molecular dynamics simulations to describe nano- to microsecond flexibility and to estimate up to millisecond kinetics of captured conformational transitions. The methodology represents the antibodies as conformational ensembles and allows comprehensive analysis of structural diversity, thermodynamics of conformations and kinetics of structural transitions. Referring to the concept of conformational selection we investigated the link between promiscuity and flexibility of the antibodies' binding interfaces. The obtained detailed characterization of the binding interface clearly indicates a link between structural flexibility and binding promiscuity for this set of antibodies

Computational Modeling and Design of Protein–Protein Interactions

Protein–protein interactions dictate biological functions, including ones essential to living organisms such as immune response or transcriptional regulation. To fundamentally understand these biological processes, we must understand the underlying interactions at the atomic scale. However interactions are overly abundant and traditional structure determination methods cannot manage a comprehensive study. Alternatively, computational methods can provide structural models with high-throughput overcoming the challenge provided by the sheer breadth of interactions, albeit at the cost of accuracy. Thus, it is necessary to improve modeling techniques if these approaches will be used to rigorously study protein–protein interactions. In this dissertation, I describe my advances to protein–protein interaction modeling (docking) methods in Rosetta. My advances are based on challenges encountered in a blind docking competition, including: modeling camelid antibodies, modeling flexible protein regions, and modeling solvated interfaces. First, I detail improvements to RosettaAntibody and Rosetta SnugDock, including making the underlying code more robust and easy to use, enabling new loop modeling methods, developing an automatically updating database, and implementing scientific benchmarks. These improvements permitted me to conduct the largest-to-date study of antibody CDR-H3 loop flexibility, which showed that traditional, small-scale studies missed emergent properties. Then, I pivot from antibodies to focus on the modeling of disordered protein regions. I contributed advances to the FloppyTail protocol, including enabling the modeling of multiple disordered regions within a single protein and pioneering an ensemble-based analysis of resultant models. I modeled Hfq proteins across six species of bacteria and demonstrated experimentally-validated prediction of interactions between disordered and ordered protein regions. My simulations provided a hypothetical mechanism for Hfq function. Finally, I designed crystallographic protein–protein interactions, with the goal of improving protein crystal resolution. To approach this exceptional challenge, I first demonstrated that, under homogenous conditions, Rosetta scores can correlate with crystal resolution. Next, I computationally designed and experimentally characterized sixteen variants of a model protein. Only five crystallized, with one providing an improvement in resolution, showing that improvement through computational design is challenging, but possible. In sum, my work advanced our understanding and our ability to model and design several challenging protein–protein interactions

Molecular characterization of public anti-PfCSP antibodies in human malaria

Plasmodium falciparum is a unicellular parasite that throughout its complex lifecycle infects Anopheles mosquitos and humans. The parasite stages injected into the human by the mosquito are called sporozoites and can be neutralized by antibodies generated by the human immune system. The present study investigates the humoral immune response, i.e. the antibody response, to the major antigen on the sporozoite surface, circumsporozoite protein (CSP). A recently published study describes high-affine anti-CSP antibodies, which are generated upon repeated controlled human malaria infection of European donors, who were never exposed to the parasite before. The present work on the one hand describes the molecular characteristics that determine the binding of such antibodies. On the other hand, it investigates whether African donors from an endemic malaria region exhibited similar antibodies. In the repertoire of the European donors, several groups of antibodies were identified that share highly similar amino acid sequences and binding behaviours. It is shown that these antibodies can only bind to the antigen if very specific sequence characteristics are conserved. This probably restricts the number of potential B cell precursors that can lead to the generation of such antibodies. The most important result of this study is the observation that antibodies binding the repetitive region of CSP directly interact with each other. The antibodies even show signs of anti-idiotope affinity maturation directed against the antigen binding site of neighbouring antibodies. Surprisingly in none of the three probed African donors antibodies with measurable anti-CSP reactivity could be found. The present work suggests that this is due to the low frequency of B cell precursors, but also due to the specific binding mode that is induced by the repetitive CSP structure

Elicitation of antibody responses against the HIV-1 gp41 Membrane Proximal External Region (MPER)

An effective vaccine to protect against HIV-1/AIDS remains elusive due to the extensive mechanisms employed by the HIV-1 virus to evade immune attack. Highly potent broadly neutralizing antibodies isolated from chronically infected individuals, however, show that such relevant antibodies can be naturally produced, implying that their elicitation through vaccination is a realistic possibility. These broadly neutralizing antibodies target different regions on the trimeric spikes formed by three protomers of the envelope (Env) protein. Each Env protein is comprised of the gp120 surface subunit in non-covalent association with the gp41 transmembrane subunit. Four regions have been identified: the CD4 binding site, the V1/V2 segment and the V3/glycan area all on the gp120 subunit as well as the MPER segment on the gp41 subunit. This dissertation focuses on the gp41 MPER segment given its highly conserved amino acid sequence among all HIV-1 clades and viral strain isolates and essential function in Env-mediated fusion and HIV entry. Of note, the MPER segment contains several adjacent epitopes targeted by broadly neutralizing antibodies, suggesting that the immune system is capable of producing neutralizing antibodies against this specific region. Analysis of both clade B and C MPER segments shows them to be L-shaped, consisting of two  helices separated by a hinge. We have found that the hinge region of the MPER segment provides the conformational flexibility necessary for the Env-mediated hemifusion and fusion processes. A significant reduction in virus infectivity is observed when the hinge region is disrupted by introduction of two amino acid mutations that eliminate -helical capping residues and the tandem hinge joints. The importance of the hinge region of the MPER segment is further supported by the action of four MPER-specific neutralizing antibodies 2F5, 4E10, 10E8 and Z13E1. These neutralizing antibodies block virus infection by disrupting MPER hinge-related function

from the immune system to neural networks

Storing memory of molecular encounters is vital for an effective response to recurring external stimuli. Interestingly, memory strategies vary among different biological processes. These strategies range from networks that process input signals and retrieve an associative memory to specialized receptors that bind only to related stimuli. The adaptive immune system uses such a specialized strategy and can provide specific responses against many pathogens. During its response, the immune system retains some cells as memory to act quicker when reinfections with the same or evolved pathogens occur. However, differentiation of memory cells remains one of the least understood cell fate decisions in immunology. The ability of immune memory to recognize evolved pathogens makes it an ideal starting point to study learning and memory strategies for evolving environments—a topic with applications far beyond immunology. In this thesis, I present three projects that study different aspects of memory strategies for evolving stimuli. Indeed, we find that specialized memory strategies can follow the evolution of stimuli and reliably recover memory of previous encounters. In contrast, fully connected networks, such as Hopfield networks, fail to reliably recover the memory of evolving stimuli. Thus, pathogen evolution might be the reason that the immune system produces specialized memories. We further find that specialized memory receptors should trade off their maximal binding for cross-reactivity to bind to evolved targets. To produce such receptors, the differentiation into memory cells in the immune system should be highly regulated. Finally, we study update strategies of memory repertoires using an energy-based model. We find that repertoires should have a moderate risk tolerance to fluctuations in performance to adapt to the evolution of targets. Nevertheless, these systems can be very efficient in distinguishing between evolved versions of stored targets and novel random stimuli.2022-01-2

Unraveling the intricacies of spatial organization of the ErbB receptors and downstream signaling pathways

Faced with the complexity of diseases such as cancer which has 1012 mutations, altering gene expression, and disrupting regulatory networks, there has been a paradigm shift in the biological sciences and what has emerged is a much more quantitative field of biology. Mathematical modeling can aid in biological discovery with the development of predictive models that provide future direction for experimentalist. In this work, I have contributed to the development of novel computational approaches which explore mechanisms of receptor aggregation and predict the effects of downstream signaling. The coupled spatial non-spatial simulation algorithm, CSNSA is a tool that I took part in developing, which implements a spatial kinetic Monte Carlo for capturing receptor interactions on the cell membrane with Gillespies stochastic simulation algorithm, SSA, for temporal cytosolic interactions. Using this framework we determine that receptor clustering significantly enhances downstream signaling. In the next study the goal was to understand mechanisms of clustering. Cytoskeletal interactions with mobile proteins are known to hinder diffusion. Using a Monte Carlo approach we simulate these interactions, determining at what cytoskeletal distribution and receptor concentration optimal clustering occurs and when it is inhibited. We investigate oligomerization induced trapping to determine mechanisms of clustering, and our results show that the cytoskeletal interactions lead to receptor clustering. After exploring the mechanisms of clustering we determine how receptor aggregation effects downstream signaling. We further proceed by implementing the adaptively coarse grained Monte Carlo, ACGMC to determine if \u27receptor-sharing\u27 occurs when receptors are clustered. In our proposed \u27receptor-sharing\u27 mechanism a cytosolic species binds with a receptor then disassociates and rebinds a neighboring receptor. We tested our hypothesis using a novel computational approach, the ACGMC, an algorithm which enables the spatial temporal evolution of the system in three dimensions by using a coarse graining approach. In this framework we are modeling EGFR reaction-diffusion events on the plasma membrane while capturing the spatial-temporal dynamics of proteins in the cytosol. From this framework we observe \u27receptor-sharing\u27 which may be an important mechanism in the regulation and overall efficiency of signal transduction. In summary, I have helped to develop predictive computational tools that take systems biology in a new direction.\u2

Addressing the Role of Mitochondrial Thioredoxin Reductase and xCT in the Maintenance of Redox Homeostasis

The thioredoxin system, along with the glutathione (GSH)-dependent system, is critically involved in the maintenance of the intracellular redox balance. The thioredoxin dependent system consists of thioredoxin reductases, thioredoxins and thioredoxin-dependent peroxidases. The mitochondrial thioredoxin reductase (Txnrd2) is an important component of the mitochondrial antioxidant system. Using Txnrd2 null cells we show that Txnrd2−/− cells produced more ROS, and were highly susceptible to different prooxiditans and GSH depletion. Administration of various antioxidants, such as NAC, GSH and α-Toc, reverted the phenotyp of the Txnrd2−/− cells. The up-regulation of the mitochondrial peroxiredoxins Prx III and Prx V and sulfiredoxin transcripts in Txnrd2−/− cells could be a compensatory mechanism for loss of Txnrd2. However, under oxidative stress, Txnrd2−/− cells showed higher amount of overoxidation of Prx I and PrxII/PrxIII indicating that in absence of Txnrd2 ROS scavenging efficiency of Trx2-Prx system is greatly compromised. Oxidative stress has been implicated in cardiovascular diseases, including infarction and heart failure. Disruption of Txnrd2 leads to perturbed heart development and embryonic lethality in mice. Heart-specific Txnrd2 disruption causes post-natal death due to biventricular dilatation of the heart and mitochondrial aberrations of cardiomyocytes. Additional evidence for a possible involvement of Txnrd2 in the pathogenesis of cardiac diseases came form the study of the patients suffering from dialated cardiomyopathy (DCM). By DNA sequence analysis of these samples, we found two novel mutations (Ala59Thr and Gly375Arg) in TXNRD2. Stable expression of murine Txnrd2 harboring these two mutations in Txnrd2−/− cells showed a dominant negative effect and were unable to rescue the cells from GSH depletion. Our data strongly suggest that the mutations found in a small percentage of DCM cases might be due to loss of Txnrd2 functions. The cellular redox balance is maintained by thioredoxin- and GSH-dependent system along with Cys/(Cys)2-cycle which is a distinct redox node of major importance. Previous study in the lab showed that the essential requirement of GSH can be bypassed by the Cys/(Cys)2-cycle. To gain further insights into the role of the thioredoxin system being a driving force for the Cys/(Cys)2-cycle, xCT was overexpressed in Txnrd2−/− fibroblasts. xCT overexpression rescued the Txnrd2−/− cells form GSH depletion. This suggests that the (Cys)2/Cys redox cycle is functional and intact even in the absence of GSH and/or mitochondrial thioredoxin reductase. In a parallel study, we found that cytosolic thioredoxin reductase (Txnrd1) is the driving force behind the Cys/(Cys)2 redox cycle. xCT epression provided growth advantages in culture conditions. In order to recapitulate our findings in vivo, we generated xCT knock-in mice, in which xCT expression can be induced in a spatio-temporal manner by tamoxifen. To our great surprise, overexpression of xCT in mice resulted in adverse effects like atrophy of spleen, thymus and testis, defective erythropoiesis and ultimately death after 5 weeks of induction. Detailed analysis of the bone marrow revealed that although there is an increase in the hematopoietic stem cell population, xCT overexpression leads to impaired erythropoiesis. The observed paradox with xCT overexpression could be due to glutamate-mediated toxicity or impaired redox balance