Chorography of blood proteins

What is considered to be representative of protein structure has changed over time, mostly driven by methodology. For a long time, proteins were characterized by their size and overall shape. After amino acid sequencing came into common use in the mid-20th century, proteins were described by the linear primary sequence of their polypeptide chains. The concept of their hierarchical structure in terms of primary, secondary, tertiary, and quaternary structures was well known, and some secondary structures were predicted. However, most representations of proteins in publications and talks at that time were a linear sequence of coded letters. When 3-dimensional structures were first determined by X-ray crystallography, graphical representations in space largely supplanted sequences. There was generally a single structure for each protein, so the view of proteins as static, rigid structures arose, with many useful structure-function relationships. In the latter part of the 20th century, scientists began to realize that the view of proteins with every atom rigidly defined in space was unrealistic from both functional and thermodynamic viewpoints. Methods to analyze protein flexibility and structural changes were developed. Structures of different conformations of individual proteins have been solved and molecular dynamics simulations have revealed a world of protein movement. It is now generally recognized that knowledge of protein dynamics is necessary for understanding protein functions.

Oddly, fibrinogen might be considered a prototypical protein since Berzelius coined the term protein in 1838 to describe a distinct class of biomolecules, saying “the name protein that I propose for the organic oxide of fibrin and albumin, I wanted to derive from the Greek word πρωτειος, because it appears to be the primitive or principal substance of animal nutrition.” [1] Fifty years ago, some protein structures had been solved but there was no consensus on the shape of fibrinogen molecules, and all kinds of crazy structures were proposed with proponents of each but only scant evidence for most of them. It was like the parable of the elephant and the blind men. Each blind person felt different parts of the elephant and came up with various contradictory ideas for the form of the elephant.

At that time, protein structure determination of fibrinogen went slowly from analysis of the amino acid sequence and arrangement of disulfide bonds to the X-ray crystallographic structure of fragments of the molecule. Fibrinogen, made of 3 pairs of polypeptide chains, with a total mass of 340 kDa, was not easy to solve at that time. Initially, low-resolution shapes were derived and then the atomic-level structure of fragments was determined. Even today, we have an X-ray crystallographic structure of only about two-thirds of the molecule, although molecular dynamics simulations have provided structural information on some of the more flexible parts of the protein [2].

The flexibility or structural dynamism of fibrinogen became apparent even during the process of crystallization and structure determination. Early crystals were made from a modified bovine fibrinogen that had been digested by a protease from contaminating bacteria, removing the αC regions, suggesting that this part of the molecule was flexible and thus interfered with crystallization [3,4]. The flexibility of fibrinogen has also been demonstrated previously by small-angle X-ray scattering, dynamic light scattering, and nuclear magnetic resonance diffusometry [2]. Flexible regions or hinges were observed in X-ray crystallographic structures and imaging by transmission electron microscopy, atomic force microscopy, and molecular dynamics simulations [2,5].

In this issue of the Journal of Thrombosis and Haemostasis, Pinelo et al. [5] present a conformationally dynamic model of fibrinogen in solution, using biophysical approaches involving 2 solution-based techniques, temperature-dependent hydrogen-deuterium exchange mass spectrometry, and small-angle X-ray scattering, corroborated by negative contrast electron microscopy. Hydrogen-deuterium exchange mass spectrometry allows the determination of regions of a protein that are buried in the protein structure and not exchangeable compared with those on the surface that are exchangeable. Hydrogen-deuterium exchange mass spectrometry studies at different temperatures further allow the identification of flexible regions of a protein. Regions that exhibit low exchange behavior that are independent of temperature represent the interior of stable or inflexible protein domains, whereas regions that exhibit high exchange behavior that are independent of temperature represent peptides that are unstructured or highly dynamic. On the other hand, regions that exhibit moderate exchange behavior that change with temperature indicate flexibility. Small-angle X-ray scattering is a method that gives curves for proteins in the solution that depend in a complex way on protein structure and flexibility.

The results in this article demonstrate a high degree of internal protein flexibility along the central scaffold of the protein with 1 hinge located within the coiled-coil connector and the other located at the interface that connects the 2 halves of the molecule. The authors proposed that the fibrinogen structure in solution consists of a complex, conformational landscape with multiple local minima [5]. They also pointed out that there are numerous point mutations associated with dysfibrinogenemias and posttranslational modifications that are near fibrinogen flexible regions. This work provides a molecular basis for the structural dynamism of fibrinogen that is likely to be important for its intermolecular interactions and for the mechanical properties of blood clots.

This article presents an example as proof of principle, in which they removed sialic acid from fibrinogen’s carbohydrate moieties and found that the X-ray scattering pattern was different, suggesting a difference in the flexibility of the molecule. There was also a difference in fibrin polymerization and clot structure, although that could arise from the removal of the sialic acid negative charge and hydrophilicity [6].

The authors of this article initially used the term chorography to describe the nature of their studies. In Ptolemy’s text of the Geographia, he defined geography as the study of the entire world, but chorography as the study of its smaller parts [7]. Even more relevant, chorography was seen as a means of knowing about places different from either geography or topography. In structural biology, if the geography of a protein corresponds to its entire topographic or atomic-level structure, chorography could represent the detailed study of smaller parts of the protein’s structure and their physical properties, such as the movement of its joints or flexible regions.

Why is the flexibility of a protein interesting or important? Proteins are like tiny molecular machines, and movement is often necessary for many of their functions, yet we still know little about the flexibility and conformational changes of most proteins. Blood proteins are subject to many forces, such as blood flow and pressure that could change the conformation of proteins. Thus, the flexibility of fibrinogen described in this article may be important for functions of soluble fibrinogen, such as its interactions with other proteins.

Fibrin and other components that makeup clots or thrombi are exposed to many more forces, such as shear forces from flowing blood at the vessel wall or plugging a hole in the wall or compressive forces on an occlusive clot [8]. Contractions of heart muscle on a thrombus in one of the chambers and the skeletal muscle surrounding veins exert compressive forces on clots within the vessel. Blood clot contraction generated by platelets pulling on fibrin exert tensile forces on fibrin fibers.

It has been long known that epitopes for antibodies or binding sites are exposed in the conversion of fibrinogen to fibrin. Cryptic binding sites in fibrinogen have been identified or suggested for plasminogen, tissue-type plasminogen activator, integrins, fibronectin, vascular endothelial cadherin, very-low-density lipoprotein, heparin, and amyloid β [5]. One likely explanation for their exposure is a change in conformation, since the fibrin molecules forming the scaffold of clots are under tension. Because of the 2-fold symmetry of the fibrinogen molecule and the locations of the complementary binding sites for polymerization, fibrin protofibrils are twisted. When they aggregate laterally, the protofibrils twist around each other. At the same time, they aggregate with 22.5-nm periodicity in register, with the result that as additional protofibrils are added to a thickening fiber, they must be stretched to accommodate the increasing path-length [9]. Consequently, fibers are under tension, stretching the fibrin molecules and likely exposing epitopes or binding sites.

Fibrinogen evolved to respond to all these forces, with specific structural changes in certain domains. When clots are exposed to sufficient force, distinct regions of fibrin molecules unfold. The exact sequence of unfolding is still being defined, but the C-terminal γ chain is involved [8]. In addition, α-helical coiled coils unfold transiently to β sheets to take up slack [8]. Thus, the α-helical coiled coil acts as a molecular spring, which represents a novel function for the coiled coil.

The movements of the hinges or flexible regions of fibrinogen described in this article that might occur under various loading regimes are as yet mostly unknown but are likely to be important. Moreover, their role in the diverse functions of fibrin(ogen) and the mechanical properties of clots or thrombi are also yet to be determined.

Many other proteins involved in thrombosis and hemostasis are subject to forces in flowing blood and some of them are part of a clot or thrombus exposed to the forces described above for fibrin. The connections between chorography and the biomechanics of thrombosis and hemostasis will be an increasingly important part of future research.